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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. T396I Mutation of Mouse Sufu Reduces the Stability and Activity of Gli3 Repressor

    PubMed Central

    Makino, Shigeru; Zhulyn, Olena; Mo, Rong; Puviindran, Vijitha; Zhang, Xiaoyun; Murata, Takuya; Fukumura, Ryutaro; Ishitsuka, Yuichi; Kotaki, Hayato; Matsumaru, Daisuke; Ishii, Shunsuke; Hui, Chi-Chung; Gondo, Yoichi

    2015-01-01

    Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396 residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3∆699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated. PMID:25760946

  3. 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

  4. 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.

  5. A Novel Gli3 Enhancer Controls the Gli3 Spatiotemporal Expression Pattern through a TALE Homeodomain Protein Binding Site ▿‡

    PubMed Central

    Coy, Sarah; Caamaño, Jorge H.; Carvajal, Jaime; Cleary, Michael L.; Borycki, Anne-Gaëlle

    2011-01-01

    The zinc finger transcription factor Gli3 is an essential mediator of hedgehog signaling. Gli3 has a dynamic expression pattern during embryonic development. In the neural tube, Gli3 transcripts are patterned along the anteroposterior and dorsoventral axes such that the initial broad expression in the posterior neural tube becomes dorsally restricted as neurogenesis takes place. Little is known about the molecular mechanisms that regulate this dynamic expression. Here, we report on a phylogenetic analysis of the Gli3 locus that uncovered a novel regulatory element, HCNE1. HCNE1 contains a compound Pbx/Meis binding site that binds Pbx and Meis/Prep proteins in vitro and in vivo. We show that HCNE1 recapitulates Gli3 expression in the developing neural tube and that mutations in the Pbx/Meis binding site affect the spatiotemporal control of HCNE1 transcriptional activity. Ectopic expression or loss of function of Pbx and Meis/Prep proteins in the chick and mouse embryo results in aberrant expression of endogenous Gli3 transcripts. We propose a novel role for TALE proteins in establishing the correct spatiotemporal expression pattern of Gli3 in the vertebrate spinal cord, thus implicating TALE transcription factors in early embryonic patterning events controlled by Sonic hedgehog signaling. PMID:21262763

  6. 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

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

    PubMed

    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. PMID:27046536

  8. GLI3 Constrains Digit Number by Controlling Both Progenitor Proliferation and BMP-Dependent Exit to Chondrogenesis

    PubMed Central

    Lopez-Rios, Javier; Speziale, Dario; Robay, Dimitri; Scotti, Martina; Osterwalder, Marco; Nusspaumer, Gretel; Galli, Antonella; Holländer, Georg A.; Kmita, Marie; Zeller, Rolf

    2015-01-01

    SUMMARY Inactivation of Gli3, a key component of Hedgehog signaling in vertebrates, results in formation of additional digits (polydactyly) during limb bud development. The analysis of mouse embryos constitutively lacking Gli3 has revealed the essential GLI3 functions in specifying the anteroposterior (AP) limb axis and digit identities. We conditionally inactivated Gli3 during mouse hand plate development, which uncoupled the resulting preaxial polydactyly from known GLI3 functions in establishing AP and digit identities. Our analysis revealed that GLI3 directly restricts the expression of regulators of the G1–S cell-cycle transition such as Cdk6 and constrains S phase entry of digit progenitors in the anterior hand plate. Furthermore, GLI3 promotes the exit of proliferating progenitors toward BMP-dependent chondrogenic differentiation by spatiotemporally restricting and terminating the expression of the BMP antagonist Gremlin1. Thus, Gli3 is a negative regulator of the proliferative expansion of digit progenitors and acts as a gatekeeper for the exit to chondrogenic differentiation. PMID:22465667

  9. 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.

  10. 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.

  11. 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.

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

    PubMed

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

    1985-05-01

    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

  13. Cross-regulation between an alternative splicing activator and a transcription repressor controls neurogenesis.

    PubMed

    Raj, Bushra; O'Hanlon, Dave; Vessey, John P; Pan, Qun; Ray, Debashish; Buckley, Noel J; Miller, Freda D; Blencowe, Benjamin J

    2011-09-01

    Neurogenesis requires the concerted action of numerous genes that are regulated at multiple levels. However, how different layers of gene regulation are coordinated to promote neurogenesis is not well understood. We show that the neural-specific Ser/Arg repeat-related protein of 100 kDa (nSR100/SRRM4) negatively regulates REST (NRSF), a transcriptional repressor of genes required for neurogenesis. nSR100 directly promotes alternative splicing of REST transcripts to produce a REST isoform (REST4) with greatly reduced repressive activity, thereby activating expression of REST targets in neural cells. Conversely, REST directly represses nSR100 in nonneural cells to prevent the activation of neural-specific splicing events. Consistent with a critical role for nSR100 in the inhibition of REST activity, blocking nSR100 expression in the developing mouse brain impairs neurogenesis. Our results thus reveal a fundamental role for direct regulatory interactions between a splicing activator and transcription repressor in the control of the multilayered regulatory programs required for neurogenesis. PMID:21884984

  14. Control of transcription of gal repressor and isorepressor genes in Escherichia coli.

    PubMed Central

    Weickert, M J; Adhya, S

    1993-01-01

    Two regulatory proteins, Gal repressor and isorepressor, control the expression of the gal and mgl operons in Escherichia coli. The transcription start sites for galR and galS, the genes for the repressor and isorepressor, were determined by primer extension of in vivo transcripts. Study of the promoter-lacZ gene fusions introduced into the chromosome indicated that galS expression was elevated in cells in which the normal galS gene was interrupted, but not in cells in which the galR gene was deleted. When both genes were disrupted, galS expression was further elevated. Expression from the galS promoter was stimulated by the addition of D-fucose, repressed by glucose, and dependent on cyclic AMP receptor protein (CRP). Expression of a similar gene fusion of the galR promoter to lacZ was unregulated. Both galR and galS genes contain two potential operator sites (OE and OI) and a CRP-binding site. The arrangement of OE, OI, and the CRP-binding site in the galS gene is analogous to the arrangement in the gal and mgl promoters, but the arrangement in galR is atypical. The increased concentration of the isorepressor when inducer is present may facilitate early shutoff of the isorepressor-regulated genes of the gal regulon when inducer (substrate) concentration falls. Images PMID:8416900

  15. Translation Repressors, an RNA Helicase, and Developmental Cues Control RNP Phase Transitions during Early Development

    PubMed Central

    Hubstenberger, Arnaud; Noble, Scott L.; Cameron, Cristiana; Evans, Thomas C.

    2013-01-01

    SUMMARY Like membranous organelles, large-scale coassembly of macromolecules can organize functions in cells. Ribonucleoproteins (RNPs) can form liquid or solid aggregates, but control and consequences of these RNP states in living, developing tissue are poorly understood. Here, we show that regulated RNP factor interactions drive transitions among diffuse, semiliquid, or solid states to modulate RNP sorting and exchange in the Caenorhabditis elegans oocyte cytoplasm. Translation repressors induce an intrinsic capacity of RNP components to coassemble into either large semiliquids or solid lattices, whereas a conserved RNA helicase prevents polymerization into nondynamic solids. Developmental cues dramatically alter both fluidity and sorting within large RNP assemblies, inducing a transition from RNP segregation in quiescent oocytes to dynamic exchange in the early embryo. Therefore, large-scale organization of gene expression extends to the cytoplasm, where regulation of supramolecular states imparts specific patterns of RNP dynamics. PMID:24176641

  16. 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.

  17. 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.

  18. Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo.

    PubMed

    Possoz, Christophe; Filipe, Sergio R; Grainge, Ian; Sherratt, David J

    2006-06-01

    We report an efficient, controllable, site-specific replication roadblock that blocks cell proliferation, but which can be rapidly and efficiently reversed, leading to recovery of viability. Escherichia coli replication forks of both polarities stalled in vivo within the first 500 bp of a 10 kb repressor-bound array of operator DNA-binding sites. Controlled release of repressor binding led to rapid restart of the blocked replication fork without the participation of homologous recombination. Cytological tracking of fork stalling and restart showed that the replisome-associated SSB protein remains associated with the blocked fork for extended periods and that duplication of the fluorescent foci associated with the blocked operator array occurs immediately after restart, thereby demonstrating a lack of sister cohesion in the region of the array. Roadblocks positioned near oriC or the dif site did not prevent replication and segregation of the rest of the chromosome.

  19. 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.

  20. Point mutations throughout the GLI3 gene cause Greig cephalopolysyndactyly syndrome.

    PubMed

    Kalff-Suske, M; Wild, A; Topp, J; Wessling, M; Jacobsen, E M; Bornholdt, D; Engel, H; Heuer, H; Aalfs, C M; Ausems, M G; Barone, R; Herzog, A; Heutink, P; Homfray, T; Gillessen-Kaesbach, G; König, R; Kunze, J; Meinecke, P; Müller, D; Rizzo, R; Strenge, S; Superti-Furga, A; Grzeschik, K H

    1999-09-01

    Greig cephalopolysyndactyly syndrome, characterized by craniofacial and limb anomalies (GCPS; MIM 175700), previously has been demonstrated to be associated with translocations as well as point mutations affecting one allele of the zinc finger gene GLI3. In addition to GCPS, Pallister-Hall syndrome (PHS; MIM 146510) and post-axial polydactyly type A (PAP-A; MIM 174200), two other disorders of human development, are caused by GLI3 mutations. In order to gain more insight into the mutational spectrum associated with a single phenotype, we report here the extension of the GLI3 mutation analysis to 24 new GCPS cases. We report the identification of 15 novel mutations present in one of the patient's GLI3 alleles. The mutations map throughout the coding gene regions. The majority are truncating mutations (nine of 15) that engender prematurely terminated protein products mostly but not exclusively N-terminally to or within the central region encoding the DNA-binding domain. Two missense and two splicing mutations mapping within the zinc finger motifs presumably also interfere with DNA binding. The five mutations identified within the protein regions C-terminal to the zinc fingers putatively affect additional functional properties of GLI3. In cell transfection experiments using fusions of the DNA-binding domain of yeast GAL4 to different segments of GLI3, transactivating capacity was assigned to two adjacent independent domains (TA(1)and TA(2)) in the C-terminal third of GLI3. Since these are the only functional domains affected by three C-terminally truncating mutations, we postulate that GCPS may be due either to haploinsufficiency resulting from the complete loss of one gene copy or to functional haploinsufficiency related to compromised properties of this transcription factor such as DNA binding and transactivation. PMID:10441342

  1. 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

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

    PubMed

    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

  3. 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

  4. miRNAs control insulin content in pancreatic β-cells via downregulation of transcriptional repressors

    PubMed Central

    Melkman-Zehavi, Tal; Oren, Roni; Kredo-Russo, Sharon; Shapira, Tirosh; Mandelbaum, Amitai D; Rivkin, Natalia; Nir, Tomer; Lennox, Kim A; Behlke, Mark A; Dor, Yuval; Hornstein, Eran

    2011-01-01

    MicroRNAs (miRNAs) were shown to be important for pancreas development, yet their roles in differentiated β-cells remain unclear. Here, we show that miRNA inactivation in β-cells of adult mice results in a striking diabetic phenotype. While islet architecture is intact and differentiation markers are maintained, Dicer1-deficient β-cells show a dramatic decrease in insulin content and insulin mRNA. As a consequence of the change in insulin content, the animals become diabetic. We provide evidence for involvement of a set of miRNAs in regulating insulin synthesis. The specific knockdown of miR-24, miR-26, miR-182 or miR-148 in cultured β-cells or in isolated primary islets downregulates insulin promoter activity and insulin mRNA levels. Further, miRNA-dependent regulation of insulin expression is associated with upregulation of transcriptional repressors, including Bhlhe22 and Sox6. Thus, miRNAs in the adult pancreas act in a new network that reinforces insulin expression by reducing the expression of insulin transcriptional repressors. PMID:21285947

  5. Coordinated control of senescence by lncRNA and a novel T-box3 co-repressor complex

    PubMed Central

    Kumar P, Pavan; Emechebe, Uchenna; Smith, Richard; Franklin, Sarah; Moore, Barry; Yandell, Mark; Lessnick, Stephen L; Moon, Anne M

    2014-01-01

    Cellular senescence is a crucial tumor suppressor mechanism. We discovered a CAPERα/TBX3 repressor complex required to prevent senescence in primary cells and mouse embryos. Critical, previously unknown roles for CAPERα in controlling cell proliferation are manifest in an obligatory interaction with TBX3 to regulate chromatin structure and repress transcription of CDKN2A-p16INK and the RB pathway. The IncRNA UCA1 is a direct target of CAPERα/TBX3 repression whose overexpression is sufficient to induce senescence. In proliferating cells, we found that hnRNPA1 binds and destabilizes CDKN2A-p16INK mRNA whereas during senescence, UCA1 sequesters hnRNPA1 and thus stabilizes CDKN2A-p16INK. Thus CAPERα/TBX3 and UCA1 constitute a coordinated, reinforcing mechanism to regulate both CDKN2A-p16INK transcription and mRNA stability. Dissociation of the CAPERα/TBX3 co-repressor during oncogenic stress activates UCA1, revealing a novel mechanism for oncogene-induced senescence. Our elucidation of CAPERα and UCA1 functions in vivo provides new insights into senescence induction, and the oncogenic and developmental properties of TBX3. DOI: http://dx.doi.org/10.7554/eLife.02805.001 PMID:24876127

  6. 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

  7. Controlled transcriptional regulation in eukaryotes by a novel transcription factor derived from Escherichia coli purine repressor.

    PubMed

    Yeon, Eun-Hee; Noh, Ju-Young; Kim, Jong-Min; Lee, Min-Young; Yoon, Sarah; Park, Sang-Kyu; Choi, Kang-Yell; Kim, Kyung-Sup

    2004-06-25

    Unlike the DNA-binding domains (DBD) of most eukaryotic transcription factors, Escherichia coli LacI family transcription factors are unable to bind to specific target DNA sequences without a cofactor-binding domain. In the present study, we reconstructed a novel DBD designated as PurHG, which binds constitutively to a 16bp purine repressor operator, by fusion of the purine repressor (PurR) DBD (residues 1-57) and the GAL4 dimerization domain (DD, residues 42-148). Binding of PurHG to DNA requires the dimerization and a hinge helix of PurR DBD. When the PurHG was expressed as a fusion protein in a form of a transcription activator (PurAD) or an artificial nuclear receptor (PurAPR or PurAER) responding to ligand, such as RU486 or beta-estradiol, it could regulate the expression of the reporter genes in NIH3T3 cells. The prerequisite region of the GAL4 DD for DNA-binding was amino acid residues from 42 to 98 in the form of PurAD, while the amino acid residues from 42 to 75 were sufficient for ligand-dependent regulation in the form of PurAPR. These results suggest that the dimerization function of the progesterone ligand-binding domain could be substituted for region 76-98 of the GAL4 DD. In summary, the fusion of the PurR DBD and the GAL4 DD generates fully active DNA-binding protein, PurHG, in vitro and in vivo, and these results provide the direct evidence of structural predictions that the proximate positioning of PurR hinge helical regions is critical for DNA-binding.

  8. Regulation of Escherichia coli purA by purine repressor, one component of a dual control mechanism.

    PubMed

    He, B; Zalkin, H

    1994-02-01

    Escherichia coli purA encodes adenylosuccinate synthetase, one of two enzymes required for synthesis of AMP from IMP. purA is subject to two- to threefold regulation by purR and about twofold regulation by a purR-independent mechanism. The 5'-flanking region of purA confers purR-dependent transcriptional regulation of purA but not the purR-independent regulation. Two operator sites in the 5'-flanking region which bind purine repressor in vitro and are required for in vivo regulation were identified. The purR-independent regulation may be posttranscriptional. It is now established that all transcription units involved in de novo synthesis of purine nucleotides, nine pur operons, as well as purR itself and guaBA, are subject to purR control.

  9. A GntR-type transcriptional repressor controls sialic acid utilization in Bifidobacterium breve UCC2003.

    PubMed

    Egan, Muireann; O'Connell Motherway, Mary; van Sinderen, Douwe

    2015-02-01

    Bifidobacterium breve strains are numerically prevalent among the gut microbiota of healthy, breast-fed infants. The metabolism of sialic acid, a ubiquitous monosaccharide in the infant and adult gut, by B. breve UCC2003 is dependent on a large gene cluster, designated the nan/nag cluster. This study describes the transcriptional regulation of the nan/nag cluster and thus sialic acid metabolism in B. breve UCC2003. Insertion mutagenesis and transcriptome analysis revealed that the nan/nag cluster is regulated by a GntR family transcriptional repressor, designated NanR. Crude cell extract of Escherichia coli EC101 in which the nanR gene had been cloned and overexpressed was shown to bind to two promoter regions within this cluster, each of which containing an imperfect inverted repeat that is believed to act as the NanR operator sequence. Formation of the DNA-NanR complex is prevented in the presence of sialic acid, which we had previously shown to induce transcription of this gene cluster.

  10. Fins into limbs: Autopod acquisition and anterior elements reduction by modifying gene networks involving 5'Hox, Gli3, and Shh.

    PubMed

    Tanaka, Mikiko

    2016-05-01

    Two major morphological changes occurred during the fin-to-limb transition: the appearance of the autopod, and the reduction of anterior skeletal elements. In the past decades, numerous approaches to the study of genetic developmental systems involved in patterning of fins/limbs among different taxa have provided clues to better understand the mechanism of the fin-to-limb transition. In this article, I discuss recent progress toward elucidating the evolutionary origin of the autopod and the mechanism through which the multiple-basal bones of ancestral fins were reduced into a single bone (humerus/femur). A particular focus of this article is the patterning mechanism of the tetrapod limb and chondrichthyan fin controlled by gene networks involving the 5'Hox genes, Gli3 and Shh. These recent data provide possible scenarios that could have led to the transformation of fins into limbs. PMID:26992366

  11. GLI3-dependent repression of DR4 mediates hedgehog antagonism of TRAIL-induced apoptosis.

    PubMed

    Kurita, S; Mott, J L; Almada, L L; Bronk, S F; Werneburg, N W; Sun, S-Y; Roberts, L R; Fernandez-Zapico, M E; Gores, G J

    2010-08-26

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through its cognate receptors death receptor 4 (DR4) and death receptor 5 (DR5), preferentially in malignant cells. However, many malignant cells remain resistant to TRAIL cytotoxicity by poorly characterized mechanisms. Here, using cholangiocarcinoma cells, as a model for TRAIL resistance, we identified a role for the oncogenic Hedgehog (Hh)-GLI pathway in the regulation of TRAIL cytotoxicity. Blockade of Hh using pharmacological and genetic tools sensitizes the cells to TRAIL cytotoxicity. Restoration of apoptosis sensitivity coincided with upregulation of DR4 expression, while expression of other death effector proteins remained unaltered. Knockdown of DR4 mimics Hh-mediated resistance to TRAIL cytotoxicity. Hh regulates the expression of DR4 by modulating the activity of its promoter. Luciferase, chromatin immunoprecipitation and expression assays show that the transcription factor GLI3 binds to the DR4 promoter and Hh requires an intact GLI3-repression activity to silence DR4 expression. Finally, small interfering RNA (siRNA)-targeted knockdown of GLI3, but not GLI1 or GLI2, restores DR4 expression and TRAIL sensitivity, indicating that the Hh effect is exclusively mediated by this transcription factor. In conclusion, these data provide evidence of a regulatory mechanism, which modulates TRAIL signaling in cancer cells and suggest new therapeutic approaches for TRAIL-resistant neoplasms. PMID:20562908

  12. GLI3-dependent repression of DR4 mediates hedgehog antagonism of TRAIL-induced apoptosis

    PubMed Central

    Kurita, S; Mott, JL; Almada, LL; Bronk, SF; Werneburg, NW; Sun, S-Y; Roberts, LR; Fernandez-Zapico, ME; Gores, GJ

    2010-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through its cognate receptors death receptor 4 (DR4) and death receptor 5 (DR5), preferentially in malignant cells. However, many malignant cells remain resistant to TRAIL cytotoxicity by poorly characterized mechanisms. Here, using cholangiocarcinoma cells, as a model for TRAIL resistance, we identified a role for the oncogenic Hedgehog (Hh)-GLI pathway in the regulation of TRAIL cytotoxicity. Blockade of Hh using pharmacological and genetic tools sensitizes the cells to TRAIL cytotoxicity. Restoration of apoptosis sensitivity coincided with upregulation of DR4 expression, while expression of other death effector proteins remained unaltered. Knockdown of DR4 mimics Hh-mediated resistance to TRAIL cytotoxicity. Hh regulates the expression of DR4 by modulating the activity of its promoter. Luciferase, chromatin immunoprecipitation and expression assays show that the transcription factor GLI3 binds to the DR4 promoter and Hh requires an intact GLI3-repression activity to silence DR4 expression. Finally, small interfering RNA (siRNA)-targeted knockdown of GLI3, but not GLI1 or GLI2, restores DR4 expression and TRAIL sensitivity, indicating that the Hh effect is exclusively mediated by this transcription factor. In conclusion, these data provide evidence of a regulatory mechanism, which modulates TRAIL signaling in cancer cells and suggest new therapeutic approaches for TRAIL-resistant neoplasms. PMID:20562908

  13. 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

  14. 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

  15. 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.

  16. Radiation abolishes inducer binding to lactose repressor.

    PubMed

    Gillard, Nathalie; Spotheim-Maurizot, Mélanie; Charlier, Michel

    2005-04-01

    The lactose operon functions under the control of the repressor-operator system. Binding of the repressor to the operator prevents the expression of the structural genes. This interaction can be destroyed by the binding of an inducer to the repressor. If ionizing radiations damage the partners, a dramatic dysfunction of the regulation system may be expected. We showed previously that gamma irradiation hinders repressor-operator binding through protein damage. Here we show that irradiation of the repressor abolishes the binding of the gratuitous inducer isopropyl-1-beta-D-thiogalactoside (IPTG) to the repressor. The observed lack of release of the repressor from the complex results from the loss of the ability of the inducer to bind to the repressor due to the destruction of the IPTG binding site. Fluorescence measurements show that both tryptophan residues located in or near the IPTG binding site are damaged. Since tryptophan damage is strongly correlated with the loss of IPTG binding ability, we conclude that it plays a critical role in the effect. A model was built that takes into account the kinetic analysis of damage production and the observed protection of its binding site by IPTG. This model satisfactorily accounts for the experimental results and allows us to understand the radiation-induced effects. PMID:15799700

  17. Gli3Xt−J/Xt−J mice exhibit lambdoid suture craniosynostosis which results from altered osteoprogenitor proliferation and differentiation

    PubMed Central

    Rice, David P.C.; Connor, Elaine C.; Veltmaat, Jacqueline M.; Lana-Elola, Eva; Veistinen, Lotta; Tanimoto, Yukiho; Bellusci, Saverio; Rice, Ritva

    2010-01-01

    Gli3 is a zinc-finger transcription factor whose activity is dependent on the level of hedgehog (Hh) ligand. Hh signaling has key roles during endochondral ossification; however, its role in intramembranous ossification is still unclear. In this study, we show that Gli3 performs a dual role in regulating both osteoprogenitor proliferation and osteoblast differentiation during intramembranous ossification. We discovered that Gli3Xt−J/Xt−J mice, which represent a Gli3-null allele, exhibit craniosynostosis of the lambdoid sutures and that this is accompanied by increased osteoprogenitor proliferation and differentiation. These cellular changes are preceded by ectopic expression of the Hh receptor Patched1 and reduced expression of the transcription factor Twist1 in the sutural mesenchyme. Twist1 is known to delay osteogenesis by binding to and inhibiting the transcription factor Runx2. We found that Runx2 expression in the lambdoid suture was altered in a pattern complimentary to that of Twist1. We therefore propose that loss of Gli3 results in a Twist1-, Runx2-dependent expansion of the sutural osteoprogenitor population as well as enhanced osteoblastic differentiation which results in a bony bridge forming between the parietal and interparietal bones. We show that FGF2 will induce Twist1, normalize osteoprogenitor proliferation and differentiation and rescue the lambdoid suture synostosis in Gli3Xt−J/Xt−J mice. Taken together, we define a novel role for Gli3 in osteoblast development; we describe the first mouse model of lambdoid suture craniosynostosis and show how craniosynostosis can be rescued in this model. PMID:20570969

  18. The lac repressor.

    PubMed

    Lewis, Mitchell

    2005-06-01

    Few proteins have had such a strong impact on a field as the lac repressor has had in Molecular Biology. Over 40 years ago, Jacob and Monod [Genetic regulatory mechanisms in the synthesis of proteins, J. Mol. Biol. 3 (1961) 318] proposed a model for gene regulation, which survives essentially unchanged in contemporary textbooks. It is a cogent depiction of how a set of 'structural' genes may be coordinately transcribed in response to environmental conditions and regulates metabolic events in the cell. In bacteria, the genes required for lactose utilization are negatively regulated when a repressor molecule binds to an upstream cis activated operator. The repressor and its operator together form a genetic switch, the lac operon. The switch functions when inducer molecules alter the conformation of the repressor in a specific manner. In the presence of a particular metabolite, the repressor undergoes a conformational change that reduces its affinity for the operator. The structures of the lac repressor and its complexes with operator DNA and effector molecules have provided a physical platform for visualizing at the molecular level the different conformations the repressor and the molecular basis for the switch. The structures of lac repressor, bound to its operator and inducer, have also been invaluable for interpreting a plethora of biochemical and genetic data. PMID:15950160

  19. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function.

    PubMed

    Haycraft, Courtney J; Banizs, Boglarka; Aydin-Son, Yesim; Zhang, Qihong; Michaud, Edward J; Yoder, Bradley K

    2005-10-01

    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. PMID:16254602

  20. 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

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

    PubMed

    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

  2. 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.

  3. 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

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

    PubMed

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

    2013-11-01

    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.

  5. Conformation of cyclo(-L-Pro-Gly-)(3) and its Ca and Mg complexes.

    PubMed

    Kartha, G; Varughese, K I; Aimoto, S

    1982-07-01

    The synthetic hexapeptide cyclo(-L-Pro-Gly-)(3) is an ionophore that shows interesting conformational changes upon binding metal ions. X-ray crystallographic studies of this peptide show that when it is crystallized from an ethanol/ethyl acetate mixture the ring takes up an asymmetric conformation containing one cis peptide bond. In crystals of a Ca(2+) complex, the cation is sandwiched between two peptide molecules that differ markedly in conformation. However, both exhibit threefold symmetric forms, with all six peptide bonds in the molecule occurring in the usual trans conformation. The Ca(2+) is octahedrally surrounded by six glycyl carbonyl oxygens from the two peptides at an average distance of 2.26 A and can easily be released by the disruption of the peptide sandwich. In the magnesium complex, the peptide forms a 1:1 complex with the ion. The Mg(2+) is octahedrally coordinated to three glycyl carbonyls and three water oxygens. The average coordination distance between magnesium and the peptide oxygens is 2.03 A and that between magnesium and water oxygen is 2.11 A. The two peptide molecules in the asymmetric unit have similar conformations and have approximate threefold symmetry.

  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. 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

  8. Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR.

    PubMed

    DeLoughery, Aaron; Dengler, Vanina; Chai, Yunrong; Losick, Richard

    2016-01-01

    Biofilm formation by Bacillus subtilis is largely governed by a circuit in which the response regulator Spo0A turns on the gene for the anti-repressor SinI. SinI, in turn, binds to and inactivates SinR, a dedicated repressor of genes for matrix production. Mutants of the genes ylbF, ymcA and yaaT are blocked in biofilm formation, but the mechanism by which they act has been mysterious. A recent report attributed their role in biofilm formation to stimulating Spo0A activity. However, we detect no measurable effect on the transcription of sinI. Instead, we find that the block in biofilm formation is caused by an increase in the levels of SinR and of its mRNA. Evidence is presented that YlbF, YmcA and YaaT interact with, and control the activity of, RNase Y, which is known to destabilize sinR mRNA. We also show that the processing of another target of RNase Y, cggR-gapA mRNA, similarly depends on YlbF and YmcA. Our work suggests that sinR mRNA stability is an additional posttranscriptional control mechanism governing the switch to multicellularity and raises the possibility that YlbF, YmcA and YaaT broadly regulate mRNA stability as part of an RNase Y-containing, multi-subunit complex.

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

    PubMed

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

    2012-05-01

    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.

  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. 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

  12. Small RNAs endow a transcriptional activator with essential repressor functions for single-tier control of a global stress regulon.

    PubMed

    Gogol, Emily B; Rhodius, Virgil A; Papenfort, Kai; Vogel, Jörg; Gross, Carol A

    2011-08-01

    The Escherichia coli σ(E) envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The σ(E) stress response was characterized as a single-tier activation network comprised of ~100 genes, including the MicA and RybB noncoding sRNAs. These highly expressed sRNAs were thought to carry out the specialized function of halting de novo synthesis of several abundant porins when envelope homeostasis was perturbed. Using a systematic target profiling and validation approach we discovered that MicA and RybB are each global mRNA repressors of both distinct and shared targets, and that the two sRNAs constitute a posttranscriptional repression arm whose regulatory scope rivals that of the protein-based σ(E) activation arm. Intriguingly, porin mRNAs constitute only ~1/3 of all targets and new nonporin targets predict roles for MicA and RybB in crosstalk with other regulatory responses. This work also provides an example of evolutionarily unrelated sRNAs that are coinduced and bind the same targets, but at different sites. Our finding that expression of either MicA or RybB sRNA protects the cell from the loss of viability experienced when σ(E) activity is inadequate illustrates the importance of the posttranscriptional repression arm of the response. σ(E) is a paradigm of a single-tier stress response with a clear division of labor in which highly expressed noncoding RNAs (MicA, RybB) endow a transcriptional factor intrinsically restricted to gene activation (σ(E)) with the opposite repressor function. PMID:21768388

  13. Small RNAs endow a transcriptional activator with essential repressor functions for single-tier control of a global stress regulon

    PubMed Central

    Gogol, Emily B.; Rhodius, Virgil A.; Papenfort, Kai; Vogel, Jörg; Gross, Carol A.

    2011-01-01

    The Escherichia coli σE envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The σE stress response was characterized as a single-tier activation network comprised of ∼100 genes, including the MicA and RybB noncoding sRNAs. These highly expressed sRNAs were thought to carry out the specialized function of halting de novo synthesis of several abundant porins when envelope homeostasis was perturbed. Using a systematic target profiling and validation approach we discovered that MicA and RybB are each global mRNA repressors of both distinct and shared targets, and that the two sRNAs constitute a posttranscriptional repression arm whose regulatory scope rivals that of the protein-based σE activation arm. Intriguingly, porin mRNAs constitute only ∼1/3 of all targets and new nonporin targets predict roles for MicA and RybB in crosstalk with other regulatory responses. This work also provides an example of evolutionarily unrelated sRNAs that are coinduced and bind the same targets, but at different sites. Our finding that expression of either MicA or RybB sRNA protects the cell from the loss of viability experienced when σE activity is inadequate illustrates the importance of the posttranscriptional repression arm of the response. σE is a paradigm of a single-tier stress response with a clear division of labor in which highly expressed noncoding RNAs (MicA, RybB) endow a transcriptional factor intrinsically restricted to gene activation (σE) with the opposite repressor function. PMID:21768388

  14. Atypical E2F transcriptional repressor DEL1 acts at the intersection of plant growth and immunity by controlling the hormone salicylic acid.

    PubMed

    Chandran, Divya; Rickert, Joshua; Huang, Yingxiang; Steinwand, Michael A; Marr, Sharon K; Wildermuth, Mary C

    2014-04-01

    In plants, the activation of immunity is often inversely correlated with growth. Mechanisms that control plant growth in the context of pathogen challenge and immunity are unclear. Investigating Arabidopsis infection with the powdery mildew fungus, we find that the Arabidopsis atypical E2F DEL1, a transcriptional repressor known to promote cell proliferation, represses accumulation of the hormone salicylic acid (SA), an established regulator of plant immunity. DEL1-deficient plants are more resistant to pathogens and slightly smaller than wild-type. The resistance and size phenotypes of DEL1-deficient plants are due to the induction of SA and activation of immunity in the absence of pathogen challenge. Moreover, Enhanced Disease Susceptibility 5 (EDS5), a SA transporter required for elevated SA and immunity, is a direct repressed target of DEL1. Together, these findings indicate that DEL1 control of SA levels contributes to regulating the balance between growth and immunity in developing leaves.

  15. 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

  16. Normal exon copy number of the GLI2 and GLI3 genes in patients with esophageal atresia.

    PubMed

    Bednarczyk, D; Smigiel, R; Patkowski, D; Laczmanska, I; Lebioda, A; Laczmanski, L; Sasiadek, M M

    2013-01-01

    Esophageal atresia (EA) is a congenital developmental defect of the alimentary tract concerning the interruption of the esophagus with or without connection to the trachea. The incidence of EA is 1 in 3000-3500 of live-born infants, and occurs in both isolated and syndromic (in combination with abnormalities in other organ systems) forms. The molecular mechanisms underlying the development of EA are poorly understood. Knockout studies in mice indicate that genes like Sonic hedgehog, Gli2, and Gli3 play a role in the etiology of EA. These facts led us to hypothesize that Sonic hedgehog-GLI gene rearrangements are associated with EA in humans. To test this hypothesis, we screened patients with isolated and syndromic EA for GLI2 and/or GLI3 microrearrangements using methods to estimate the copy number (Multiplex Ligation-dependent Probe Amplification, real-time polymerase chain reaction). To our best knowledge this is the first study assessing copy number of GLI2 and GLI3 genes in patients with EA. PMID:23442119

  17. Centrosomal Protein DZIP1 Regulates Hedgehog Signaling by Promoting Cytoplasmic Retention of Transcription Factor GLI3 and Affecting Ciliogenesis*

    PubMed Central

    Wang, Chengbing; Low, Wee-Chuang; Liu, Aimin; Wang, Baolin

    2013-01-01

    The primary cilium is required for Hedgehog signaling. So far, all known ciliogenic proteins regulate Hedgehog signaling through their role in ciliogenesis. Here we show that the mouse DZIP1 regulates Hedgehog signaling through two mechanisms. First, DZIP1 interacts with GLI3, a transcriptional regulator for Hedgehog signaling, and prevents GLI3 from entering the nucleus. Second, DZIP1 is required for ciliogenesis. We show that DZIP1 colocalizes and interacts with CEP164, a protein localizing at appendages of the mother centrioles, and IFT88, a component of the intraflagellar transport (IFT) machinery. Functionally, both CEP164 and Ninein appendage proteins fail to localize to ciliary appendages in Dzip1 mutant cells; IFT components are not recruited to the basal body of cilia. Importantly, the accumulation of GLI3 in the nucleus is independent of loss of primary cilia in Dzip1 mutant cells. Therefore, DZIP1 is the first known ciliogenic protein that regulates Hedgehog signaling through a dual mechanism and that biochemically links IFT machinery with Hedgehog pathway components. PMID:23955340

  18. Natural repressors of P-induced hybrid dysgenesis in Drosophila melanogaster: a model for repressor evolution.

    PubMed

    Corish, P; Black, D M; Featherston, D W; Merriam, J; Dover, G A

    1996-04-01

    Type I repressors control P element transposition and comprise full length elements and elements with small 3' deletions in the final exon. Using a sensitive assay for measuring the strength of repression of P element transposition in somatic and germline tissues, we have isolated and characterized a naturally occurring type I repressor element from a Q population of Drosophila melanogaster. We demonstrate that the almost complete repression of transposition in this population is a mixture of KP elements with intermediate levels of repression, and the strong contribution of a single 2.6 kb P element deletion derivative, which we call SR (Strong Repressor). A deletion in the final intron of SR allows for the constitutive production of a putative 75 kDa repressor protein in germline tissues in addition to the production of the 66 kDa repressor in the soma, which would result in a biparental mode of inheritance of repression. Based on the four observed classes of natural Q populations, we propose a model in which populations containing SR-like elements, capable of producing strong type I repressor constitutively, have a selective advantage over populations which rely either on maternally transmitted P cytotype or on KP-induced weak levels of repression. Such populations may subsequently spread and constitute an evolutionary stable strategy for the repression of hybrid dysgenesis in Drosophila melanogaster. PMID:8801184

  19. Natural repressors of P-induced hybrid dysgenesis in Drosophila melanogaster: a model for repressor evolution.

    PubMed

    Corish, P; Black, D M; Featherston, D W; Merriam, J; Dover, G A

    1996-04-01

    Type I repressors control P element transposition and comprise full length elements and elements with small 3' deletions in the final exon. Using a sensitive assay for measuring the strength of repression of P element transposition in somatic and germline tissues, we have isolated and characterized a naturally occurring type I repressor element from a Q population of Drosophila melanogaster. We demonstrate that the almost complete repression of transposition in this population is a mixture of KP elements with intermediate levels of repression, and the strong contribution of a single 2.6 kb P element deletion derivative, which we call SR (Strong Repressor). A deletion in the final intron of SR allows for the constitutive production of a putative 75 kDa repressor protein in germline tissues in addition to the production of the 66 kDa repressor in the soma, which would result in a biparental mode of inheritance of repression. Based on the four observed classes of natural Q populations, we propose a model in which populations containing SR-like elements, capable of producing strong type I repressor constitutively, have a selective advantage over populations which rely either on maternally transmitted P cytotype or on KP-induced weak levels of repression. Such populations may subsequently spread and constitute an evolutionary stable strategy for the repression of hybrid dysgenesis in Drosophila melanogaster.

  20. 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

  1. 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

  2. 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

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

    PubMed

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

    2016-09-01

    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

  4. Studies on gene control regions X. The effect of specific adenine-thymine transversions on the lac repressor-lac operator interaction.

    PubMed Central

    Sista, H S; Loder, R T; Caruthers, M H

    1979-01-01

    Chemical and enzymatic methods were used to synthesize a transition (AT to GC) and a transversion (AT to TA) at a lac operator site known to interact with lac repressor through the thymine 5 methyl group. These operators also contained a poly(dA) . poly(dT) tail 8 to 12 base pairs in length at one end. Results suggest that the steric constraints of lac repressor relative to the position of the 5 methyl group are quite critical. For example a seven fold reduction in stability was observed for the transversion. Results also suggest that the operator spans at least 21 base pairs. Images PMID:379824

  5. Response to copper stress in Streptomyces lividans extends beyond genes under direct control of a copper-sensitive operon repressor protein (CsoR).

    PubMed

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

    2012-05-18

    A copper-sensitive operon repressor protein (CsoR) has been identified in Streptomyces lividans (CsoR(Sl)) and found to regulate copper homeostasis with attomolar affinity for Cu(I). Solution studies reveal apo- and Cu(I)-CsoR(Sl) to be a tetramer assembly, and a 1.7-Å resolution crystal structure of apo-CsoR(Sl) 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 P(1)-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.

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

    PubMed

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

    2013-01-01

    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. PMID:23143103

  7. 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

  8. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26510468

  10. 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. PMID:21392509

  11. 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

  12. 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.

  13. 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.

  14. Activated RecA protein may induce expression of a gene that is not controlled by the LexA repressor and whose function is required for mutagenesis and repair of UV-irradiated bacteriophage lambda

    SciTech Connect

    Calsou, P.; Villaverde, A.; Defais, M.

    1987-10-01

    The activated form of the RecA protein (RecA) is known to be involved in the reactivation and mutagenesis of UV-irradiated bacteriophage lambda and in the expression of the SOS response in Escherichia coli K-12. The expression of the SOS response requires cleavage of the LexA repressor by RecA and the subsequent expression of LexA-controlled genes. The evidence presented here suggests that RecA induces the expression of a gene(s) that is not under LexA control and that is also necessary for maximal repair and mutagenesis of damaged phage. This conclusion is based on the chloramphenicol sensitivity of RecA -dependent repair and mutagenesis of damaged bacteriophage lambda in lexA(Def) hosts.

  15. 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

  16. STM sustains stem cell function in the Arabidopsis shoot apical meristem and controls KNOX gene expression independently of the transcriptional repressor AS1.

    PubMed

    Scofield, Simon; Dewitte, Walter; Murray, James Ah

    2014-04-28

    The Arabidopsis KNOX gene SHOOT MERISTEMLESS (STM) is required for both the development and the sustained function of the shoot apical meristem (SAM) and can induce de novo meristem formation when expressed ectopically. STM acts through induction of cytokinin (CK) synthesis to inhibit cellular differentiation and additionally functions to organize undifferentiated cells into a self-sustaining meristem. STM has been shown to positively regulate the related KNOX genes KNAT1/BP and KNAT2, and it has been proposed that this is mediated through repression of the ARP-type transcriptional repressor ASYMMETRIC LEAVES1 (AS1). Here we investigate the role of STM in SAM organization, stem cell maintenance and the regulation of KNOX gene expression. We show that culture of stm mutant explants in high CK conditions does not restore proper sustained shoot growth, supporting the idea of STM having CK-independent roles in meristem function. Furthermore, we show that STM is required for continued stem cell function in the SAM by sustaining expression of the stem cell-promoting factor WUS and preventing cells of the meristem organizing center from adopting lateral organ-specific fates. We also demonstrate that transcriptional activation of class-1 KNOX genes by STM is independent of AS1, since AS1 transcript levels are not reduced in response to STM and STM is able to transactivate expression of both KNAT1/BP and KNAT2 in the as1 mutant background.

  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. Identification of the Bacillus subtilis pur operon repressor.

    PubMed

    Weng, M; Nagy, P L; Zalkin, H

    1995-08-01

    Transcription of the Bacillus subtilis pur operon is repressed in response to a signal of excess adenine. We have purified the repressor protein and have identified, cloned, and overexpressed the purR regulatory gene that controls transcription initiation of the operon. B. subtilis purR encodes a 62-kDa homodimer that binds to the pur operon control region. The PurR binding site which overlaps the promoter encompasses approximately 110 bp. The protein-DNA interaction is inhibited by 5-phosphoribosyl 1-pyrophosphate. A mutation that deletes the repressor binding site or one that disrupts purR abolishes binding activity in vitro and repression of transcription in vivo in response to the excess adenine signal. These results lead to a model in which an excess-adenine signal is transmitted to PurR via the 5-phosphoribosyl 1-pyrophosphate pool. In addition, purR is autoregulated. There is no structural or mechanistic similarity between the B. subtilis and Escherichia coli purine repressors.

  19. Interaction of Bacillus subtilis purine repressor with DNA.

    PubMed

    Shin, B S; Stein, A; Zalkin, H

    1997-12-01

    A purine repressor (PurR) mediates adenine nucleotide-dependent regulation of transcription initiation of the Bacillus subtilis pur operon. This repressor has been purified for the first time, and binding to control site DNA was characterized. PurR binds in vitro to four operons. Apparent Kd values for binding were 7 nM for the pur operon, 8 nM for purA, 13 nM for purR, and 44 nM for the pyr operon. In each case, DNase I footprints exhibited a pattern of protected and hypersensitive sites that extended over more than 60 bp. A GAAC-N24-GTTC sequence in the pur operon was necessary but not sufficient for the PurR-DNA interaction. However, this motif, which is conserved in the four binding sites, was not required for binding of PurR to purA. Thus, the common DNA recognition element for binding of PurR to the four operons is not known. Multiple PurR-pur operon DNA complexes having a binding stoichiometry that was either approximately two or six repressor molecules per DNA fragment were detected. The results of a torsional constraint experiment suggest that control site DNA forms one right-handed turn around PurR.

  20. 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.

  1. 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

    2016-01-01

    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

  2. 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

  3. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25908840

  5. 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

  6. Transcriptional co-repressors and memory storage

    PubMed Central

    Schoch, Hannah; Abel, Ted

    2014-01-01

    Epigenetic modifications are a central mechanism for regulating chromatin structure and gene expression in the brain. A wide array of histone- and DNA-modifying enzymes have been identified as critical regulators of neuronal function, memory formation, and as causative agents in neurodevelopmental and neuropsychiatric disorders. Chromatin modifying enzymes are frequently incorporated into large multi-protein co-activator and co-repressor complexes, where the activity of multiple enzymes is both spatially and temporally coordinated. In this review, we discuss negative regulation of gene expression by corepressor complexes, and the role of co-repressors and their binding partners in neuronal function, memory, and disease. PMID:24440532

  7. Timing is everything in plant development. The central role of floral repressors.

    PubMed

    Jarillo, Jose A; Piñeiro, Manuel

    2011-10-01

    Progress in understanding the molecular basis of flowering time control has revealed that floral repressors play a central role in modulating the floral transition and are essential to prevent the precocious onset of flowering. A number of cellular processes including chromatin remodeling, selective protein degradation, and transcriptional regulation mediated by transcription factors are involved in repressing the initiation of flowering. Floral repressors interact at different levels with floral inductive pathways and prevent the premature onset of flowering that could impact negatively on the reproductive success of plants. Despite recent advances, further studies will be needed to understand how the interactions between floral repressors and the regulatory networks involved in the control of flowering time have evolved in different species. Recent data suggest that a diversity of regulatory proteins act as central floral repressors in different plants, and even in those species where regulatory modules are conserved new elements that modulate the function of these pathways have been recruited to mediate specific adaptive responses. The development of genomic tools and predictive models that can integrate large datasets related to the flowering behavior of plant species will facilitate the characterization of the repressor mechanisms underlying flowering responses, a trait with implications in the yield of crop species. In a scenario of global climate change, an in depth understanding of these gene circuits will be essential for the development of crop varieties with improved yield.

  8. 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. PMID:23005801

  9. 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.

  10. 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.

  11. 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.

  12. 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. PMID:27223409

  13. Characterization of the interaction of the glp repressor of Escherichia coli K-12 with single and tandem glp operator variants.

    PubMed Central

    Zhao, N; Oh, W; Trybul, D; Thrasher, K S; Kingsbury, T J; Larson, T J

    1994-01-01

    The glp operons of Escherichia coli are negatively controlled by the glp repressor. Comparison of the repressor-binding affinities for consensus and altered consensus operators in vivo showed that all base substitutions at positions 3, 4, 5, and 8 from the center of the palindromic operator caused a striking decrease in repressor binding. Substitutions at other positions had a severe to no effect on repressor binding, depending on the base substitution. The results obtained indicate that the repressor binds with highest affinity to operators with the half-site WATKYTCGWW, where W is A or T, K is G or T, and Y is C or T. Strong cooperative binding of the repressor to tandem operators was demonstrated in vivo. Cooperativity was maximal when two 20-bp operators were directly repeated or when 2 bp separated the two operators. Cooperativity decreased with the deletion of 2 bp or the addition of 4 bp between the individual operators. Cooperativity was eliminated with a 6-bp insertion between the operators. PMID:8157609

  14. 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

  15. 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. PMID:23818216

  16. Decoding transcriptional repressor complexes in the adult central nervous system.

    PubMed

    Adachi, Megumi; Monteggia, Lisa M

    2014-05-01

    Cells maintain precise gene expression by balancing transcriptional activation and repression. While much work has focused on elucidating transcriptional activation in the central nervous system (CNS), little is known about transcriptional repression. One means to repress gene expression is to initiate binding of transcription factors to DNA, which then recruit co-repressors as well as other accessory proteins, forming a multi-protein repressor complex. These multi-protein repressor complexes include histone modifying enzymes that trigger processes such as histone acetylation, methylation, and ubiquitylation, altering chromatin structures to impact gene expression. Within these complexes transcriptional repressor proteins per se do not exhibit enzymatic reactions to remodel chromatin structure, whereas histone modifying enzymes lack intrinsic DNA binding activity but have an ability to process post-translational modifications on histones. Thus, the mutual association between transcriptional repressors and histone modifying enzymes is essential to sculpt chromatin to favor transcriptional repression and down regulate gene expression. Additionally, co-repressors are integral components in the context of gene repression as they bridge the association of transcriptional repressors and histone modifying enzymes. In this review, we will discuss the roles of some of the major components of these repressor complex in the CNS as well as their cellular functions that may underlie fundamental behavior in animals.

  17. 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

  18. 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.

  19. The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding.

    PubMed

    Wilson, C J; Zhan, H; Swint-Kruse, L; Matthews, K S

    2007-01-01

    In 1961, Jacob and Monod proposed the operon model for gene regulation based on metabolism of lactose in Escherichia coli. This proposal was followed by an explication of allosteric behavior by Monod and colleagues. The operon model rationally depicted how genetic mechanisms can control metabolic events in response to environmental stimuli via coordinated transcription of a set of genes with related function (e.g. metabolism of lactose). The allosteric response found in the lactose repressor and many other proteins has been extended to a variety of cellular signaling pathways in all organisms. These two models have shaped our view of modern molecular biology and captivated the attention of a surprisingly broad range of scientists. More recently, the lactose repressor monomer was used as a model system for experimental and theoretical explorations of protein folding mechanisms. Thus, the lac system continues to advance our molecular understanding of genetic control and the relationship between sequence, structure and function. PMID:17103112

  20. Ion effects on the lac repressor--operator equilibrium.

    PubMed

    Barkley, M D; Lewis, P A; Sullivan, G E

    1981-06-23

    The effects of ions on the interaction of lac repressor protein and operator DNA have been studied by the membrane filter technique. The equilibrium association constant was determined as a function of monovalent and divalent cation concentrations, anions, and pH. The binding of repressor and operator is extremely sensitive to the ionic environment. The dependence of the observed equilibrium constant on salt concentration is analyzed according to the binding theory of Record et al. [Record, M. T., Jr., Lohman, T. M., & deHaseth, P. L. (1976) J. Mol. Biol. 107, 145]. The number of ionic interactions in repressor--operator complex is deduced from the slopes of the linear log-log plots. About 11 ionic interactions are formed between repressor and DNA phosphates at pH 7.4 and about 9 ionic interactions at pH 8.0, in reasonable agreement with previous estimates. A favorable nonelectrostatic binding free energy of about 9-12 kcal/mol is estimated from the extrapolated equilibrium constants at the 1 M standard state. The values are in good accord with recent results for the salt-independent binding of repressor core and operator DNA. The effects of pH on the repressor--operator interaction are small, and probably result from titration of functional groups in the DNA-binding site of the protein. For monovalent salts, the equilibrium constant is slightly dependent on cation type and highly dependent on anion type. At constant salt concentration, the equilibrium constant decreases about 10000-fold in the order CH3CO2- greater than or equal to F- greater than Cl- greater than Br- greater than NO3- greater than SCN- greater than I-. The wide range of accessible equilibrium constants provides a useful tool for in vitro studies of the repressor--operator interaction.

  1. The qa repressor gene of Neurospora crassa: wild-type and mutant nucleotide sequences.

    PubMed Central

    Huiet, L; Giles, N H

    1986-01-01

    The qa-1S gene, one of two regulatory genes in the qa gene cluster of Neurospora crassa, encodes the qa repressor. The qa-1S gene together with the qa-1F gene, which encodes the qa activator protein, control the expression of all seven qa genes, including those encoding the inducible enzymes responsible for the utilization of quinic acid as a carbon source. The nucleotide sequence of the qa-1S gene and its flanking regions has been determined. The deduced coding sequence for the qa-1S protein encodes 918 amino acids with a calculated molecular weight of 100,650 and is interrupted by a single 66-base-pair intervening sequence. Both constitutive and noninducible mutants occur in the qa-1S gene and two different mutations of each type have been cloned and sequenced. All four mutations occur within the predicted coding region of the qa-1S gene. This result strongly supports the hypothesis that the qa-1S gene encodes a repressor. All four mutations are located within codons for the last 300 amino acids of the qa-1S protein. The mutations in three of the mutants involve amino acid substitutions, while the fourth mutant, which has a constitutive phenotype, contains a frameshift mutation. The two constitutive mutations occur in the most distal region of the gene, possibly implicating the COOH-terminal region of the qa repressor in binding to its target. The two noninducible mutations occur in a region proximal to the constitutive mutations, possibly implicating this region of the qa repressor in binding the inducer. Images PMID:3010294

  2. Gene model 129 (Gm129) encodes a novel transcriptional repressor that modulates circadian gene expression.

    PubMed

    Annayev, Yunus; Adar, Sheera; Chiou, Yi-Ying; Lieb, Jason D; Sancar, Aziz; Ye, Rui

    2014-02-21

    The mammalian circadian clock is a molecular oscillator composed of a feedback loop that involves transcriptional activators CLOCK and BMAL1, and repressors Cryptochrome (CRY) and Period (PER). Here we show that a direct CLOCK·BMAL1 target gene, Gm129, is a novel regulator of the feedback loop. ChIP analysis revealed that the CLOCK·BMAL1·CRY1 complex strongly occupies the promoter region of Gm129. Both mRNA and protein levels of GM129 exhibit high amplitude circadian oscillations in mouse liver, and Gm129 gene encodes a nuclear-localized protein that directly interacts with BMAL1 and represses CLOCK·BMAL1 activity. In vitro and in vivo protein-DNA interaction results demonstrate that, like CRY1, GM129 functions as a repressor by binding to the CLOCK·BMAL1 complex on DNA. Although Gm129(-/-) or Cry1(-/-) Gm129(-/-) mice retain a robust circadian rhythm, the peaks of Nr1d1 and Dbp mRNAs in liver exhibit a significant phase delay compared with control. Our results suggest that, in addition to CRYs and PERs, the GM129 protein contributes to the transcriptional feedback loop by modulating CLOCK·BMAL1 activity as a transcriptional repressor.

  3. Phosphorylation of trihelix transcriptional repressor ASR3 by MAP KINASE4 negatively regulates Arabidopsis immunity.

    PubMed

    Li, Bo; Jiang, Shan; Yu, Xiao; Cheng, Cheng; Chen, Sixue; Cheng, Yanbing; Yuan, Joshua S; Jiang, Daohong; He, Ping; Shan, Libo

    2015-03-01

    Proper control of immune-related gene expression is crucial for the host to launch an effective defense response. Perception of microbe-associated molecular patterns (MAMPs) induces rapid and profound transcriptional reprogramming via unclear mechanisms. Here, we show that ASR3 (ARABIDOPSIS SH4-RELATED3) functions as a transcriptional repressor and plays a negative role in regulating pattern-triggered immunity (PTI) in Arabidopsis thaliana. ASR3 belongs to a plant-specific trihelix transcription factor family for which functional studies are lacking. MAMP treatments induce rapid phosphorylation of ASR3 at threonine 189 via MPK4, a mitogen-activated protein kinase that negatively regulates PTI responses downstream of multiple MAMP receptors. ASR3 possesses transcriptional repressor activity via its ERF-associated amphiphilic repression motifs and negatively regulates a large subset of flg22-induced genes. Phosphorylation of ASR3 by MPK4 enhances its DNA binding activity to suppress gene expression. Importantly, the asr3 mutant shows enhanced disease resistance to virulent bacterial pathogen infection, whereas transgenic plants overexpressing the wild-type or phospho-mimetic form of ASR3 exhibit compromised PTI responses. Our studies reveal a function of the trihelix transcription factors in plant innate immunity and provide evidence that ASR3 functions as a transcriptional repressor regulated by MAMP-activated MPK4 to fine-tune plant immune gene expression.

  4. 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.

  5. Structures of ω repressors bound to direct and inverted DNA repeats explain modulation of transcription

    PubMed Central

    Weihofen, Wilhelm Andreas; Cicek, Aslan; Pratto, Florencia; Alonso, Juan Carlos; Saenger, Wolfram

    2006-01-01

    Repressor ω regulates transcription of genes required for copy number control, accurate segregation and stable maintenance of inc18 plasmids hosted by Gram-positive bacteria. ω belongs to homodimeric ribbon-helix-helix (RHH2) repressors typified by a central, antiparallel β-sheet for DNA major groove binding. Homodimeric ω2 binds cooperatively to promotors with 7 to 10 consecutive non-palindromic DNA heptad repeats (5′-A/TATCACA/T-3′, symbolized by →) in palindromic inverted, converging (→←) or diverging (←→) orientation and also, unique to ω2 and contrasting other RHH2 repressors, to non-palindromic direct (→→) repeats. Here we investigate with crystal structures how ω2 binds specifically to heptads in minimal operators with (→→) and (→←) repeats. Since the pseudo-2-fold axis relating the monomers in ω2 passes the central C–G base pair of each heptad with ∼0.3 Å downstream offset, the separation between the pseudo-2-fold axes is exactly 7 bp in (→→), ∼0.6 Å shorter in (→←) but would be ∼0.6 Å longer in (←→). These variations grade interactions between adjacent ω2 and explain modulations in cooperative binding affinity of ω2 to operators with different heptad orientations. PMID:16528102

  6. Dynamic multidrug recognition by multidrug transcriptional repressor LmrR.

    PubMed

    Takeuchi, Koh; Tokunaga, Yuji; Imai, Misaki; Takahashi, Hideo; Shimada, Ichio

    2014-11-18

    LmrR is a multidrug transcriptional repressor that controls the expression of a major multidrug transporter, LmrCD, in Lactococcus lactis. However, the molecular mechanism by which LmrR binds to structurally unrelated compounds and is released from the promoter region remains largely unknown. Here, we structurally and dynamically characterized LmrR in the apo, compound-bound and promoter-bound states. The compound-binding site of LmrR exhibits ps-μs dynamics in the apo state, and compound ligation shifts the preexisting conformational equilibrium to varying extents to achieve multidrug recognition. Meanwhile, the compound binding induces redistribution of ps-ns dynamics to the allosteric sites, which entropically favors the high-affinity recognition. Furthermore, the reciprocal compound/promoter binding by LmrR is achieved by the incompatible conformational ensembles between the compound- and promoter-bound states. Collectively, the data show how LmrR can dynamically exert its functions through promiscuous multi-target interactions, in a manner that cannot be understood by a static structural view.

  7. 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

  8. 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

    2016-01-01

    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

  9. 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

    2016-01-01

    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.

  10. A cyclic carbo-isosteric penta-depsipeptide: cyclo(Phe1–d-Ala2–Gly3–Phe4–APO5)

    PubMed Central

    Guéret, Stéphanie M.; Wagner, Trixie

    2015-01-01

    The title compound, cyclo(Phe1–d-Ala2–Gly3–Phe4–APO5), C26H32N4O5, is the minor diastereoisomer of a cyclic penta-peptidomimetic analogue containing a novel 2-amino­propyl lactone (APO) motif, which displays the same number of atoms as the native amino acid glycine and has a methyl group in place of the carbonyl O atom. The crystal structure presented here allows the analysis of the secondary structure of this unprecedented cyclic carbo-isosteric depsipeptide. The conformation of the central ring is stabilized by an intra­molecular N—H⋯O hydrogen bond between the carbonyl O atom of the first residue (Phe1) and the amide group H atom of the fourth residue (Phe4). Based on the previously reported hydrogen bond and on the values of the torsion angles ϕ and ψ, the loop formed by the first, second, third and fourth residues (Phe1, d-Ala2, Gly3 and Phe4) can be classified as a type II′ β-turn. The loop around the new peptidomimetic motif, on the other hand, resembles an open γ-turn containing a weak N—H⋯O hydrogen bond between the carbonyl group O atom of the fourth residue (Phe4) and the amide unit H atom of the first residue (Phe1). In the crystal, the peptidomimetic mol­ecules are arranged in chains along the b-axis direction. Within such a chain, the mol­ecules of the structure are linked via N—H⋯O hydrogen bonds between the amide group H atom of the secondary residue (d-Ala2) and the carb­oxy unit O atom of the fourth residue (Phe4) in a neighboring mol­ecule. The newly formed methyl stereocentre of the APO peptidomimetic motif (APO5) was obtained as the minor diastereoisomer in a ring-closing reductive amination reaction and adopts an R configuration. PMID:25705467

  11. 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.

  12. An Unusual Phage Repressor Encoded by Mycobacteriophage BPs.

    PubMed

    Villanueva, Valerie M; Oldfield, Lauren M; Hatfull, Graham F

    2015-01-01

    Temperate bacteriophages express transcription repressors that maintain lysogeny by down-regulating lytic promoters and confer superinfection immunity. Repressor regulation is critical to the outcome of infection-lysogenic or lytic growth-as well as prophage induction into lytic replication. Mycobacteriophage BPs and its relatives use an unusual integration-dependent immunity system in which the phage attachment site (attP) is located within the repressor gene (33) such that site-specific integration leads to synthesis of a prophage-encoded product (gp33103) that is 33 residues shorter at its C-terminus than the virally-encoded protein (gp33136). However, the shorter form of the repressor (gp33103) is stable and active in repression of the early lytic promoter PR, whereas the longer virally-encoded form (gp33136) is inactive due to targeted degradation via a C-terminal ssrA-like tag. We show here that both forms of the repressor bind similarly to the 33-34 intergenic regulatory region, and that BPs gp33103 is a tetramer in solution. The BPs gp33103 repressor binds to five regulatory regions spanning the BPs genome, and regulates four promoters including the early lytic promoter, PR. BPs gp33103 has a complex pattern of DNA recognition in which a full operator binding site contains two half sites separated by a variable spacer, and BPs gp33103 induces a DNA bend at the full operator site but not a half site. The operator site structure is unusual in that one half site corresponds to a 12 bp palindrome identified previously, but the other half site is a highly variable variant of the palindrome. PMID:26332854

  13. 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

  14. Does the repressor coping style predict lower posttraumatic stress symptoms?

    PubMed

    McNally, Richard J; Hatch, John P; Cedillos, Elizabeth M; Luethcke, Cynthia A; Baker, Monty T; Peterson, Alan L; Litz, Brett T

    2011-07-01

    We tested whether a continuous measure of repressor coping style predicted lower posttraumatic stress disorder (PTSD) symptoms in 122 health care professionals serving in Operation Iraqi Freedom. Zero-order correlational analyses indicated that predeployment repressor coping scores negatively predicted postdeployment PTSD symptoms, r(s) = -0.29, p = 0.001, whereas predeployment Connor-Davidson Resilience Scale (CD-RISC) scores did not predict postdeployment PTSD symptoms, r(s) = -0.13, p = 0.14. However, predeployment trait anxiety was chiefly responsible for the association between repressor coping and PTSD symptom severity, r(s) = 0.38, p = 0.001. Four percent of the subjects qualified for a probable PTSD diagnosis. Although service members with relatively higher PTSD scores had lower repressor coping scores than did the other subjects, their level of predeployment anxiety was chiefly responsible for this relationship. Knowing someone's predeployment level of trait anxiety permits better prediction of PTSD symptoms among trauma-exposed service members than does knowing his or her level of repressive coping. PMID:22128715

  15. Optimized expression and purification of biophysical quantities of Lac repressor and Lac repressor regulatory domain.

    PubMed

    Stetz, Matthew A; Carter, Marie V; Wand, A Joshua

    2016-07-01

    The recombinant production of Lac repressor (LacI) in Escherichia coli is complicated by its ubiquitous use as a regulatory element in commercially-available expression vectors and host strains. While LacI-regulated expression systems are often used to produce recombinant LacI, the product can be heterogeneous and unsuitable for some studies. Alternative approaches include using unregulated vectors which typically suffer from low yield or vectors with promoters induced by metabolically active sugars which can dilute isotope labels necessary for certain biophysical studies. Here, an optimized expression system and isolation protocol for producing various constructs of LacI is introduced which eliminates these complications. The expression vector is an adaptation of the pASK backbone wherein expression of the lacI gene is regulated by an anhydrotetracyline inducible tetA promoter and the host strain lacks the lacI gene. Typical yields in highly deuterated minimal medium are nearly 2-fold greater than those previously reported. Notably, the new expression system is also able to produce the isolated regulatory domain of LacI without co-expression of the full-length protein and without any defects in cell viability, eliminating the inconvenient requirement for strict monitoring of cell densities during pre-culturing. Typical yields in highly deuterated minimal medium are significantly greater than those previously reported. Characterization by solution NMR shows that LacI constructs produced using this expression system are highly homogenous and functionally active. PMID:27064119

  16. An evolutionary link between sporulation and prophage induction in the structure of a repressor:anti-repressor complex.

    PubMed

    Lewis, R J; Brannigan, J A; Offen, W A; Smith, I; Wilkinson, A J

    1998-11-13

    Spore formation is an extreme response of some bacteria to adversity. In Bacillus subtilis the proteins of the sin, sporulation inhibition, region form a component of an elaborate molecular circuitry that regulates the commitment to sporulation. SinR is a tetrameric repressor protein that binds to the promoters of genes essential for entry into sporulation and prevents their transcription. This repression is overcome through the activity of SinI, which disrupts the SinR tetramer through the formation of a SinI-SinR heterodimer. The interactions governing this curious quaternary transition are revealed in the crystal structure of the SinI-SinR complex. The most striking, and unexpected, finding is that the tertiary structure of the DNA-binding domain of SinR is identical with that of the corresponding domains of the repressor proteins, CI and Cro, of bacteriophage 434 that regulate lysis/lysogeny. This structural similarity greatly exceeds that between SinR and any bacterial protein or between the 434 repressor proteins and their homologues in the closely related bacteriophage lambda. The close evolutionary relationship implied by the structures of SinR and the 434 repressors provokes both comparison of their functions and a speculative consideration of the intriguing possibility of an evolutionary link between the two adaptive responses, sporulation and prophage induction.

  17. Interactions between Escherichia coli RNA polymerase and lambda repressor. Mutations in PRM affect repression of PR.

    PubMed

    Hwang, J J; Gussin, G N

    1988-04-20

    The rightward operator, OR, of bacteriophage lambda is part of a complex regulatory region that includes PRM, the promoter for repressor synthesis by a prophage, the rightward early promoter PR, and three repressor-binding sites, OR1, OR2 and OR3. By binding to OR2, repressor blocks transcription from PR and simultaneously stimulates the formation of open complexes between RNA polymerase and PRM. In this letter, we describe a test of the hypothesis that the interaction between RNA polymerase bound at PRM and repressor bound at OR2 increases the apparent affinity of repressor for OR. One implication of this hypothesis is that the amount of repressor required for repression of PR should be inversely correlated with PRM promoter strength. This is indeed the case. The amount of repressor required for 50% repression of PR is decreased by prmup-1, an "up" mutation of PRM, and is increased by prm- mutations. An unexpected finding is that in addition to their effect on the apparent affinity of repressor for OR, mutations in the -35 region of PRM alter the shape of repressor-titration curves. We propose that these mutations alter the interaction between RNA polymerase bound at PRM and repressor bound at OR2 in such a way that cooperativity in the binding of repressor to OR1 and OR2 is also disrupted.

  18. FLC, a repressor of flowering, is regulated by genes in different inductive pathways.

    PubMed

    Rouse, Dean T; Sheldon, Candice C; Bagnall, David J; Peacock, W James; Dennis, Elizabeth S

    2002-01-01

    The MADS-box protein encoded by FLOWERING LOCUS C (FLC) is a repressor of flowering. Loci in the autonomous flowering pathway control FLC levels. We show the epistatic groupings of autonomous pathway mutants fca/fy and fve/fpa, based on their effects on flowering time, are consistent with their effects on FLC transcript and protein levels. We demonstrate that synergistic increases in FLC mRNA and protein expression occur in response to interactions between the autonomous pathway mutants fca and fpa and mutants in other pathways (fe, ft, fha) that do not regulate FLC when present as single mutants. These changes in FLC levels provide the molecular basis of the interactions previously shown in genetic analyses. The interactions between genes of multiple pathways emphasize the central position of FLC in the control of floral initiation. FLC protein levels match those of its mRNA for a range of genetic, developmental and environmental variables, indicating that control of FLC is at the level of transcription or transcript stability. The autonomous and photoperiod pathways also interact at the level of SOC1. FLC acts as a repressor of SOC1, and SOC1 levels are low when FLC levels are high. In C24 plants which have moderately high FLC levels, flowering occurs without a decrease in FLC level, but the SOC1 level does increase. Thus SOC1 levels can be upregulated through the activities of other pathways, despite the repression by FLC.

  19. 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.

  20. Mutations in the Bacillus subtilis purine repressor that perturb PRPP effector function in vitro and in vivo.

    PubMed

    Weng, M; Zalkin, H

    2000-07-01

    The Bacillus subtilis pur operon repressor (PurR) has a PRPP (5-phosphoribosyl 1-pyrophosphate) binding motif at residues 199-211. Two PurR PRPP binding region mutations (D203A and D204A) were constructed, and the effects on binding of repressor to the pur operon control site in vitro and on regulation of pur operon expression in vivo were investigated. PRPP significantly inhibited the binding of wild-type but not mutant PurR to pur operon control site DNA. In strains with the D203A and D204A mutations, pur operon expression in vivo was super-repressed by addition of adenine to the growth medium. These results support the role of PRPP in modulating the regulatory function of PurR in vivo. YabJ, the product of the distal gene in the bicistronic purR operon, is also required for PurR function in vivo.

  1. 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.

  2. 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. PMID:26865637

  3. 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.

  4. The TetR Family of Transcriptional Repressors

    PubMed Central

    Ramos, Juan L.; Martínez-Bueno, Manuel; Molina-Henares, Antonio J.; Terán, Wilson; Watanabe, Kazuya; Zhang, Xiaodong; Gallegos, María Trinidad; Brennan, Richard; Tobes, Raquel

    2005-01-01

    We have developed a general profile for the proteins of the TetR family of repressors. The stretch that best defines the profile of this family is made up of 47 amino acid residues that correspond to the helix-turn-helix DNA binding motif and adjacent regions in the three-dimensional structures of TetR, QacR, CprB, and EthR, four family members for which the function and three-dimensional structure are known. We have detected a set of 2,353 nonredundant proteins belonging to this family by screening genome and protein databases with the TetR profile. Proteins of the TetR family have been found in 115 genera of gram-positive, α-, β-, and γ-proteobacteria, cyanobacteria, and archaea. The set of genes they regulate is known for 85 out of the 2,353 members of the family. These proteins are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity. The regulatory network in which the family member is involved can be simple, as in TetR (i.e., TetR bound to the target operator represses tetA transcription and is released in the presence of tetracycline), or more complex, involving a series of regulatory cascades in which either the expression of the TetR family member is modulated by another regulator or the TetR family member triggers a cell response to react to environmental insults. Based on what has been learned from the cocrystals of TetR and QacR with their target operators and from their three-dimensional structures in the absence and in the presence of ligands, and based on multialignment analyses of the conserved stretch of 47 amino acids in the 2,353 TetR family members, two groups of residues have been identified. One group includes highly conserved positions involved in the proper orientation of the helix-turn-helix motif and hence seems to play a structural role. The other set of

  5. Regulated expression of a repressor protein: FadR activates iclR.

    PubMed

    Gui, L; Sunnarborg, A; LaPorte, D C

    1996-08-01

    The control of the glyoxylate bypass operon (aceBAK) of Escherichia coli is mediated by two regulatory proteins, IclMR and FadR. IclMR is a repressor protein which has previously been shown to bind to a site which overlaps the aceBAK promoter. FAR is a repressor/activator protein which participates in control of the genes of fatty acid metabolism. A sequence just upstream of the iclR promoter bears a striking resemblance to FadR binding sites found in the fatty acid metabolic genes. The in vitro binding specificity of FadR, determined by oligonucleotide selection, was in good agreement with the sequences of these sites. The ability of FadR to bind to the site associated with iclR was demonstrated by gel shift and DNase I footprint analyses. Disruption of FadR or inactivation of the FadR binding site of iclR decreased the expression of an iclR::lacZ operon fusion, indicating that FadR activates the expression of iclR. It has been reported that disruption of fadR increases the expression of aceBAK. We observed a similar increase when we inactivated the FadR binding site of an iclR+ allele. This result suggests that FadR regulates aceBAK indirectly by altering the expression of IclR. PMID:8755903

  6. Functional and Structural Analysis of HrcA Repressor Protein from Caulobacter crescentus

    PubMed Central

    Susin, Michelle F.; Perez, Humberto R.; Baldini, Regina L.; Gomes, Suely L.

    2004-01-01

    A large number of bacteria regulate chaperone gene expression during heat shock by the HrcA-CIRCE system, in which the DNA element called CIRCE serves as binding site for the repressor protein HrcA under nonstress conditions. In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction is controlled by a σ32-dependent promoter and the HrcA-CIRCE system plays a role in regulation of groESL expression under physiological temperatures. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein, and specific binding to the CIRCE element was obtained by gel shift assays. The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that the GroE chaperonin machine modulates HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells, producing different amounts of GroES/GroEL. In addition, we identified the putative DNA-binding domain of HrcA through extensive protein sequence comparison and constructed various HrcA mutant proteins containing single amino acid substitutions in or near this region. In vitro and in vivo experiments with these mutated proteins indicated several amino acids important for repressor activity. PMID:15466027

  7. 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.

  8. Myeloid Translocation Gene-16 Co-Repressor Promotes Degradation of Hypoxia-Inducible Factor 1

    PubMed Central

    Kumar, Parveen; Gullberg, Urban; Olsson, Inge; Ajore, Ram

    2015-01-01

    The myeloid translocation gene 16 (MTG16) co-repressor down regulates expression of multiple glycolytic genes, which are targets of the hypoxia-inducible factor 1 (HIF1) heterodimer transcription factor that is composed of oxygen-regulated labile HIF1α and stable HIF1β subunits. For this reason, we investigated whether MTG16 might regulate HIF1 negatively contributing to inhibition of glycolysis and stimulation of mitochondrial respiration. A doxycycline Tet-On system was used to control levels of MTG16 in B-lymphoblastic Raji cells. Results from co-association studies revealed MTG16 to interact with HIF1α. The co-association required intact N-terminal MTG16 residues including Nervy Homology Region 1 (NHR1). Furthermore, electrophoretic mobility shift assays demonstrated an association of MTG16 with hypoxia response elements (HREs) in PFKFB3, PFKFB4 and PDK1 promoters in-vitro. Results from chromatin immunoprecipitation assays revealed co-occupancy of these and other glycolytic gene promoters by HIF1α, HIF1β and MTG16 in agreement with possible involvement of these proteins in regulation of glycolytic target genes. In addition, MTG16 interacted with prolyl hydroxylase D2 and promoted ubiquitination and proteasomal degradation of HIF1α. Our findings broaden the area of MTG co-repressor functions and reveal MTG16 to be part of a protein complex that controls the levels of HIF1α. PMID:25974097

  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. 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.

  11. The Krüppel-associated box repressor domain can induce reversible heterochromatization of a mouse locus in vivo.

    PubMed

    Groner, Anna C; Tschopp, Patrick; Challet, Ludivine; Dietrich, Jens-Erik; Verp, Sonia; Offner, Sandra; Barde, Isabelle; Rodriguez, Ivan; Hiiragi, Takashi; Trono, Didier

    2012-07-20

    The study of chromatin and its regulators is key to understanding and manipulating transcription. We previously exploited the Krüppel-associated box (KRAB) transcriptional repressor domain, present in hundreds of vertebrate-specific zinc finger proteins, to assess the effect of its binding to gene bodies. These experiments revealed that the ectopic and doxycycline (dox)-controlled tet repressor KRAB fusion protein (tTRKRAB) can induce reversible and long-range silencing of cellular promoters. Here, we extend this system to in vivo applications and use tTRKRAB to achieve externally controllable repression of an endogenous mouse locus. We employed lentiviral-mediated transgenesis with promoterless TetO-containing gene traps to engineer a mouse line where the endogenous kinesin family member 2A (Kif2A) promoter drives a YFP reporter gene. When these mice were crossed to animals expressing the TetO-binding tTRKRAB repressor, this regulator was recruited to the Kif2A locus, and YFP expression was reduced. This effect was reversed when dox was given to embryos or adult mice, demonstrating that the cellular Kif2A promoter was only silenced upon repressor binding. Molecular analyses confirmed that tTRKRAB induced transcriptional repression through the spread of H3K9me3-containing heterochromatin, without DNA methylation of the trapped Kif2A promoter. Therefore, we demonstrate that targeting of tTRKRAB to a gene body in vivo results in reversible transcriptional repression through the spreading of facultative heterochromatin. This finding not only sheds light on KRAB-mediated transcriptional processes, but also suggests approaches for the externally controllable and reversible modulation of chromatin and transcription in vivo. PMID:22605343

  12. 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

  13. The protein kinase Akt1 regulates the interferon response through phosphorylation of the transcriptional repressor EMSY.

    PubMed

    Ezell, Scott A; Polytarchou, Christos; Hatziapostolou, Maria; Guo, Ailan; Sanidas, Ioannis; Bihani, Teeru; Comb, Michael J; Sourvinos, George; Tsichlis, Philip N

    2012-03-01

    The protein kinases Akt1, Akt2, and Akt3 possess nonredundant signaling properties, few of which have been investigated. Here, we present evidence for an Akt1-dependent pathway that controls interferon (IFN)-regulated gene expression and antiviral immunity. The target of this pathway is EMSY, an oncogenic interacting partner of BRCA2 that functions as a transcriptional repressor. Overexpression of EMSY in hTERT-immortalized mammary epithelial cells, and in breast and ovarian carcinoma cell lines, represses IFN-stimulated genes (ISGs) in a BRCA2-dependent manner, whereas its knockdown has the opposite effect. EMSY binds to the promoters of ISGs, suggesting that EMSY functions as a direct transcriptional repressor. Akt1, but not Akt2, phosphorylates EMSY at Ser209, relieving EMSY-mediated ISG repression. The Akt1/EMSY/ISG pathway is activated by both viral infection and IFN, and it inhibits the replication of HSV-1 and vesicular stomatitis virus (VSV). Collectively, these data define an Akt1-dependent pathway that contributes to the full activation of ISGs by relieving their repression by EMSY and BRCA2.

  14. 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

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

    PubMed Central

    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. PMID:24026510

  16. Tight-binding repressors of the lac operon: selection system and in vitro analysis.

    PubMed

    Pfahl, M

    1979-01-01

    The isolation and characterization of altered repressors of the lac operon which have an increased affinity for an operator should give useful clues about the molecular basis for the very tight and specific interaction between repressor and operator. A selection system has been devised which allows the isolation of such repressor mutants. This system selects for mutant repressors which can overcome lac operator-constitutive (Oc) mutations. By using in vivo assays, 24 candidates were obtained which, compared with wild type, have an increased trans effect of their repressor on one or several Oc operators. Three of these candidates have been investigated in vitro; the affinity of their repressor for inducer was unchanged, whereas the affinity for wild-type operator was increased 15-, 86-, and 262-fold, respectively. PMID:104955

  17. 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.

  18. 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.

  19. Transcriptional regulation of repressor synthesis in mycobacteriophage L5.

    PubMed

    Nesbit, C E; Levin, M E; Donnelly-Wu, M K; Hatfull, G F

    1995-09-01

    Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. Lysogeny is maintained by the putative repressor, the gene 71 product, which also mediates immunity to superinfection. We show here that there are three promoters located upstream of gene 71 which are active in an L5 lysogen but which do not require any phage-encoded proteins. In early lytic growth, gene 71 is also transcribed from a promoter, Pleft, located at the right end of the genome and which appears to be a target of gp71 regulation. A model is given for the regulation of L5 life cycles. PMID:8594325

  20. 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

  1. 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.

  2. MLX Is a Transcriptional Repressor of the Mammalian Golgi Stress Response.

    PubMed

    Taniguchi, Mai; Sasaki-Osugi, Kanae; Oku, Masaya; Sawaguchi, Shogo; Tanakura, Soichiro; Kawai, Yumeto; Wakabayashi, Sadao; Yoshida, Hiderou

    2016-07-30

    The Golgi stress response is a homeostatic mechanism that controls the capacity of the Golgi apparatus in accordance with cellular demands. When the capacity of the Golgi apparatus becomes insufficient (Golgi stress), transcription levels of Golgi-related genes encoding glycosylation enzymes, a Golgi structural protein, and components of vesicular transport are upregulated through a common cis-acting enhancer-the Golgi apparatus stress response element (GASE). Here, we identified the transcription factor MLX as a GASE-binding protein. MLX resides in the cytoplasm and does not bind to GASE in normal growth conditions, whereas MLX translocates into the nucleus and specifically binds to GASE in response to Golgi stress. Suppression of MLX expression increased transcriptional induction of target genes of the Golgi stress response, whereas overexpression of MLX reduced GASE-binding of TFE3 as well as transcriptional induction from GASE, suggesting that MLX is a transcriptional repressor of the mammalian Golgi stress response.

  3. Ligand interactions with lactose repressor protein and the repressor-operator complex: the effects of ionization and oligomerization on binding.

    PubMed

    Wilson, Corey J; Zhan, Hongli; Swint-Kruse, Liskin; Matthews, Kathleen S

    2007-03-01

    Specific interactions between proteins and ligands that modify their functions are crucial in biology. Here, we examine sugars that bind the lactose repressor protein (LacI) and modify repressor affinity for operator DNA using isothermal titration calorimetry and equilibrium DNA binding experiments. High affinity binding of the commonly-used inducer isopropyl-beta,D-thiogalactoside is strongly driven by enthalpic forces, whereas inducer 2-phenylethyl-beta,D-galactoside has weaker affinity with low enthalpic contributions. Perturbing the dimer interface with either pH or oligomeric state shows that weak inducer binding is sensitive to changes in this distant region. Effects of the neutral compound o-nitrophenyl-beta,D-galactoside are sensitive to oligomerization, and at elevated pH this compound converts to an anti-inducer ligand with slightly enhanced enthalpic contributions to the binding energy. Anti-inducer o-nitrophenyl-beta,D-fucoside exhibits slightly enhanced affinity and increased enthalpic contributions at elevated pH. Collectively, these results both demonstrate the range of energetic consequences that occur with LacI binding to structurally-similar ligands and expand our insight into the link between effector binding and structural changes at the subunit interface. PMID:16860458

  4. 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.

  5. REST is a hypoxia-responsive transcriptional repressor.

    PubMed

    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

    2016-08-17

    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.

  6. Purification and characterization of the diphtheria toxin repressor.

    PubMed Central

    Schmitt, M P; Twiddy, E M; Holmes, R K

    1992-01-01

    The diphtheria toxin repressor gene (dtxR) encodes a protein (DtxR) that regulates transcription of the diphtheria toxin gene (tox) by an iron-dependent mechanism. Cloned dtxR was expressed in Escherichia coli from the phage T7 gene 10 promoter, and DtxR was purified. Specific binding of DtxR to the tox+ operator was dependent on reduction of DtxR and the presence of ferrous ions. DtxR protected a sequence of approximately 30 nucleotide pairs, partially overlapping the tox promoter and containing a region of dyad symmetry, from digestion by DNase I. DtxR exhibited very little binding to the mutant tox-201 operator region and failed to bind to the promoter/operator region of the ferric uptake regulation (fur) gene of E. coli. Images PMID:1502169

  7. Tolerance of Arc repressor to multiple-alanine substitutions.

    PubMed

    Brown, B M; Sauer, R T

    1999-03-01

    Arc repressor mutants containing from three to 15 multiple-alanine substitutions have spectral properties expected for native Arc proteins, form heterodimers with wild-type Arc, denature cooperatively with Tms equal to or greater than wild type, and, in some cases, fold as much as 30-fold faster and unfold as much as 50-fold slower than wild type. Two of the mutants, containing a total of 14 different substitutions, also footprint operator DNA in vitro. The stability of some of the proteins with multiple-alanine mutations is significantly greater than that predicted from the sum of the single substitutions, suggesting that a subset of the wild-type residues in Arc may interact in an unfavorable fashion. Overall, these results show that almost half of the residues in Arc can be replaced by alanine en masse without compromising the ability of this small, homodimeric protein to fold into a stable, native-like structure. PMID:10051581

  8. Binding of the N-terminal domain of the lactococcal bacteriophage TP901-1 CI repressor to its target DNA: a crystallography, small angle scattering, and nuclear magnetic resonance study.

    PubMed

    Frandsen, Kristian H; Rasmussen, Kim K; Jensen, Malene Ringkjøbing; Hammer, Karin; Pedersen, Margit; Poulsen, Jens-Christian N; Arleth, Lise; Lo Leggio, Leila

    2013-10-01

    In most temperate bacteriophages, regulation of the choice of lysogenic or lytic life cycle is controlled by a CI repressor protein. Inhibition of transcription is dependent on a helix-turn-helix motif, often located in the N-terminal domain (NTD), which binds to specific DNA sequences (operator sites). Here the crystal structure of the NTD of the CI repressor from phage TP901-1 has been determined at 1.6 Å resolution, and at 2.6 Å resolution in complex with a 9 bp double-stranded DNA fragment that constitutes a half-site of the OL operator. This N-terminal construct, comprising residues 2-74 of the CI repressor, is monomeric in solution as shown by nuclear magnetic resonance (NMR), small angle X-ray scattering, and gel filtration and is monomeric in the crystal structures. The binding interface between the NTD and the half-site in the crystal is very similar to the interface that can be mapped by NMR in solution with a full palindromic site. The interactions seen in the complexes (in the crystal and in solution) explain the observed affinity for the OR site that is lower than that for the OL site and the specificity for the recognized DNA sequence in comparison to that for other repressors. Compared with many well-studied phage repressor systems, the NTD from TP901-1 CI has a longer extended scaffolding helix that, interestingly, is strongly conserved in putative repressors of Gram-positive pathogens. On the basis of sequence comparisons, we suggest that these bacteria also possess repressor/antirepressor systems similar to that found in phage TP901-1. PMID:24047404

  9. The tryptophan repressor sequence is highly conserved among the Enterobacteriaceae.

    PubMed Central

    Arvidson, D N; Arvidson, C G; Lawson, C L; Miner, J; Adams, C; Youderian, P

    1994-01-01

    Tryptophan biosynthesis in Escherichia coli is regulated by the product of the trpR gene, the tryptophan (Trp) repressor. Trp aporepressor binds the corepressor, L-tryptophan, to form a holorepressor complex, which binds trp operator DNA tightly, and inhibits transcription of the tryptophan biosynthetic operon. The conservation of trp operator sequences among enteric Gram-negative bacteria suggests that trpR genes from other bacterial species can be cloned by complementation in E. coli. To clone trpR homologues, a deletion of the E. coli trpR gene, delta trpR504, was made on a plasmid by site-directed mutagenesis, then crossed onto the E. coli genome. Plasmid clones of the trpR genes of Enterobacter aerogenes and Enterobacter cloacae were isolated by complementation of the delta trpR504 allele, scored as the ability to repress beta-galactosidase synthesis from a prophage-borne trpE-lacZ gene fusion. The predicted amino acid sequences of four enteric TrpR proteins show differences, clustered on the backside of the folded repressor, opposite the DNA-binding helix-turn-helix substructures. These differences are predicted to have little effect on the interactions of the aporepressor with tryptophan, holorepressor with operator DNA, or tandemly bound holorepressor dimers with one another. Although there is some variation observed at the dimer interface, interactions predicted to stabilize the interface are conserved. The phylogenetic relationships revealed by the TrpR amino acid sequence alignment agree with the results of others. PMID:8208606

  10. 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.

  11. Fur-type transcriptional repressors and metal homeostasis in the cyanobacterium Synechococcus sp. PCC 7002

    PubMed Central

    Ludwig, Marcus; Chua, Tiing Tiing; Chew, Chyue Yie; Bryant, Donald A.

    2015-01-01

    Metal homeostasis is a crucial cellular function for nearly all organisms. Some heavy metals (e.g., Fe, Zn, Co, Mo) are essential because they serve as cofactors for enzymes or metalloproteins, and chlorophototrophs such as cyanobacteria have an especially high demand for iron. At excessive levels, however, metals become toxic to cyanobacteria. Therefore, a tight control mechanism is essential for metal homeostasis. Metal homeostasis in microorganisms comprises two elements: metal acquisition from the environment and detoxification or excretion of excess metal ions. Different families of metal-sensing regulators exist in cyanobacteria and each addresses a more or less specific set of target genes. In this study the regulons of three Fur-type and two ArsR-SmtB-type regulators were investigated in a comparative approach in the cyanobacterium Synechococcus sp. PCC 7002. One Fur-type regulator controls genes for iron acquisition (Fur); one controls genes for zinc acquisition (Zur); and the third controls two genes involved in oxidative stress (Per). Compared to other well-investigated cyanobacterial strains, however, the set of target genes for each regulator is relatively small. Target genes for the two ArsR-SmtB transcriptional repressors (SmtB (SYNPCC7002_A2564) and SYNPCC7002_A0590) are involved in zinc homeostasis in addition to Zur. Their target genes, however, are less specific for zinc and point to roles in a broader heavy metal detoxification response. PMID:26582412

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

    PubMed

    Goffinont, S; Davidkova, M; Spotheim-Maurizot, 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. PMID:19520056

  13. Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli.

    PubMed

    Velkov, Tony; Jones, Alun; Lim, Maria L R

    2008-01-01

    A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni(2+)-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor. PMID:18800304

  14. Harnessing the master transcriptional repressor REST to reciprocally regulate neurogenesis

    PubMed Central

    Nesti, Edmund

    2015-01-01

    Neurogenesis begins in embryonic development and continues at a reduced rate into adulthood in vertebrate species, yet the signaling cascades regulating this process remain poorly understood. Plasma membrane-initiated signaling cascades regulate neurogenesis via downstream pathways including components of the transcriptional machinery. A nuclear factor that temporally regulates neurogenesis by repressing neuronal differentiation is the repressor element 1 (RE1) silencing transcription (REST) factor. We have recently discovered a regulatory site on REST that serves as a molecular switch for neuronal differentiation. Specifically, C-terminal domain small phosphatase 1, CTDSP1, present in non-neuronal cells, maintains REST activity by dephosphorylating this site. Reciprocally, extracellular signal-regulated kinase, ERK, activated by growth factor signaling in neural progenitors, and peptidylprolyl cis/trans isomerase Pin1, decrease REST activity through phosphorylation-dependent degradation. Our findings further resolve the mechanism for temporal regulation of REST and terminal neuronal differentiation. They also provide new potential therapeutic targets to enhance neuronal regeneration after injury. PMID:27535341

  15. Heat-Induced Fibrillation of BclXL Apoptotic Repressor

    PubMed Central

    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

    2013-01-01

    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. PMID:23714425

  16. REST is a hypoxia-responsive transcriptional repressor.

    PubMed

    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

  17. 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. PMID:23714425

  18. 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

  19. MAFG is a transcriptional repressor of bile acid synthesis and metabolism.

    PubMed

    de Aguiar Vallim, Thomas Q; Tarling, Elizabeth J; Ahn, Hannah; Hagey, Lee R; Romanoski, Casey E; Lee, Richard G; Graham, Mark J; Motohashi, Hozumi; Yamamoto, Masayuki; Edwards, Peter A

    2015-02-01

    Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis, and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG(+/-) mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR.

  20. MAFG Is a Transcriptional Repressor of Bile Acid Synthesis and Metabolism

    PubMed Central

    de Aguiar Vallim, Thomas Q.; Tarling, Elizabeth J.; Ahn, Hannah; Hagey, Lee R.; Romanoski, Casey E.; Lee, Richard G.; Graham, Mark J.; Motohashi, Hozumi; Yamamoto, Masayuki; Edwards, Peter A.

    2015-01-01

    Summary Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway, and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG+/− mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-Seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR. PMID:25651182

  1. Kcnip1 a Ca²⁺-dependent transcriptional repressor regulates the size of the neural plate in Xenopus.

    PubMed

    Néant, Isabelle; Mellström, Britt; Gonzalez, Paz; Naranjo, Jose R; Moreau, Marc; Leclerc, Catherine

    2015-09-01

    In amphibian embryos, our previous work has demonstrated that calcium transients occurring in the dorsal ectoderm at the onset of gastrulation are necessary and sufficient to engage the ectodermal cells into a neural fate by inducing neural specific genes. Some of these genes are direct targets of calcium. Here we search for a direct transcriptional mechanism by which calcium signals are acting. The only known mechanism responsible for a direct action of calcium on gene transcription involves an EF-hand Ca²⁺ binding protein which belongs to a group of four proteins (Kcnip1 to 4). Kcnip protein can act in a Ca²⁺-dependent manner as a transcriptional repressor by binding to a specific DNA sequence, the Downstream Regulatory Element (DRE) site. In Xenopus, among the four kcnips, we show that only kcnip1 is timely and spatially present in the presumptive neural territories and is able to bind DRE sites in a Ca²⁺-dependent manner. The loss of function of kcnip1 results in the expansion of the neural plate through an increased proliferation of neural progenitors. Later on, this leads to an impairment in the development of anterior neural structures. We propose that, in the embryo, at the onset of neurogenesis Kcnip1 is the Ca²⁺-dependent transcriptional repressor that controls the size of the neural plate. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

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

    PubMed

    Kasajima, Ichiro; Sasaki, Katsutomo

    2016-05-01

    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

  3. Anaphase promoting complex–dependent degradation of transcriptional repressors Nrm1 and Yhp1 in Saccharomyces cerevisiae

    PubMed Central

    Ostapenko, Denis; Solomon, Mark J.

    2011-01-01

    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 APCCdh1 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. PMID:21562221

  4. Hepatic oxidative stress activates the Gadd45b gene via degradation of the transcriptional repressor STAT3

    PubMed Central

    Kim, Jung-Hwan; Qu, Aijuan; Reddy, Janardan K.; Gao, Bin; Gonzalez, Frank J.

    2013-01-01

    Growth arrest and DNA damage-inducible beta (GADD45b) plays an important role in many intracellular events, such as cell cycle arrest, DNA repair, cell survival, apoptosis and senescence. However, its mechanism of transcriptional regulation remains unclear. In this study, the mechanism of proliferator-activated receptor α (PPARα) ligand induction of the Gadd45b gene in mouse liver was investigated. Gadd45b mRNA was markedly induced by the PPARα agonist, Wy-14,643, in wild-type mice but not in Ppara-null mice. STAT3 was found to be a repressor of the Gadd45b gene through binding to upstream regulatory elements. The role of STAT3 in control of Gadd45b was confirmed using liver-specific Stat3-null mice. Wy-14,643 treatment stimulated STAT3 ubiquitination leading to activation of the Gadd45b gene as a result of loss of Gadd45b repression by STAT3. STAT3 degradation was induced by forced overexpression of the PPARα target gene-encoded enzyme ACOX1, that produces increased H2O2 as a by product of fatty acid β-oxidation. H2O2 also stimulated expression of Gadd45b in cultured cells. These studies revealed that PPARα indirectly induces the Gadd45b gene in liver through promoting degradation of the repressor STAT3 as a result of elevated oxidative stress. PMID:23939942

  5. 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.

  6. TBLR1 regulates the expression of nuclear hormone receptor co-repressors

    PubMed Central

    Zhang, Xin-Min; Chang, Qing; Zeng, Lin; Gu, Judy; Brown, Stuart; Basch, Ross S

    2006-01-01

    Background Transcription is regulated by a complex interaction of activators and repressors. The effectors of repression are large multimeric complexes which contain both the repressor proteins that bind to transcription factors and a number of co-repressors that actually mediate transcriptional silencing either by inhibiting the basal transcription machinery or by recruiting chromatin-modifying enzymes. Results TBLR1 [GenBank: NM024665] is a co-repressor of nuclear hormone transcription factors. A single highly conserved gene encodes a small family of protein molecules. Different isoforms are produced by differential exon utilization. Although the ORF of the predominant form contains only 1545 bp, the human gene occupies ~200 kb of genomic DNA on chromosome 3q and contains 16 exons. The genomic sequence overlaps with the putative DC42 [GenBank: NM030921] locus. The murine homologue is structurally similar and is also located on Chromosome 3. TBLR1 is closely related (79% homology at the mRNA level) to TBL1X and TBL1Y, which are located on Chromosomes X and Y. The expression of TBLR1 overlaps but is distinct from that of TBL1. An alternatively spliced form of TBLR1 has been demonstrated in human material and it too has an unique pattern of expression. TBLR1 and the homologous genes interact with proteins that regulate the nuclear hormone receptor family of transcription factors. In resting cells TBLR1 is primarily cytoplasmic but after perturbation the protein translocates to the nucleus. TBLR1 co-precipitates with SMRT, a co-repressor of nuclear hormone receptors, and co-precipitates in complexes immunoprecipitated by antiserum to HDAC3. Cells engineered to over express either TBLR1 or N- and C-terminal deletion variants, have elevated levels of endogenous N-CoR. Co-transfection of TBLR1 and SMRT results in increased expression of SMRT. This co-repressor undergoes ubiquitin-mediated degradation and we suggest that the stabilization of the co-repressors by TBLR1

  7. Frameshift mutations in the bacteriophage Mu repressor gene can confer a trans-dominant virulent phenotype to the phage.

    PubMed Central

    Geuskens, V; Vogel, J L; Grimaud, R; Desmet, L; Higgins, N P; Toussaint, A

    1991-01-01

    Virulent mutations in the bacteriophage Mu repressor gene were isolated and characterized. Recombination and DNA sequence analysis have revealed that virulence is due to unusual frameshift mutations which change several C-terminal amino acids. The vir mutations are in the same repressor region as the sts amber mutations which, by eliminating several C-terminal amino acids, suppress thermosensitivity of repressor binding to the operators by its N-terminal domain (J. L. Vogel, N. P. Higgins, L. Desmet, V. Geuskens, and A. Toussaint, unpublished data). Vir repressors bind Mu operators very poorly. Thus the Mu repressor C terminus, either by itself or in conjunction with other phage or host proteins, tunes the DNA-binding properties at the repressor N terminus. Images FIG. 3 FIG. 4 FIG. 5 PMID:1833383

  8. SLUG is a Direct Transcriptional Repressor of PTEN Tumor Suppressor

    PubMed Central

    Uygur, Berna; Abramo, Katrina; Leikina, Evgenia; Vary, Calvin; Liaw, Lucy; Wu, Wen-Shu

    2015-01-01

    BACGORUND PTEN/AKT signaling plays a key role in prostate cancer development and maintenance of prostate cancer stem cells. How other oncogenes or tumor suppressors interact with this pathway remain to be elucidated. SLUG is an zinc finger transcription factor of the Snail superfamily, and it promotes cancer metastasis and determines the mammary stem cell state. METHODS SLUG was overexpressed in cells by retroviral vector and knockdown of SLUG and PTEN was mediated by shRNAs-expressing lentiviruses. Expression level of SLUG and PTEN was examined by Western blot, RT-PCR, and qPCR analyses. PTEN promoter activity was measured by luciferase reporter assay. ChIP assay was used to measure the binding between SLUG and the PTEN promoter in vivo. RESULT We showed that overexpression of SLUG decreased expression of PTEN tumor repressor in prostate cancer cell lines 22RV1 and DU145; conversely, knockdown of SLUG expression elevated PTEN expresson at both protein and RNA level in these cells. We demonstrated that SLUG overexpression inhibits PTEN promoter activity through the proximal promoter region in prostate cancer cells. By ChIP assay, we confirmed that SLUG directly binds to the PTEN promoter region covering the E-box sites. We also showed that Slug deficiency leads to an increased expression of PTEN in mouse embryo fibroblasts and prostate tissues. Importantly, we found that overexpression of SLUG increases drug resistance of DU145 prostate cancer cell line and knockdown of SLUG by shRNA sensitizes DU145 cell line to chemotherapeutic drugs. We further demonstrated that PTEN knockdown converts drug sensitivity of DU145 cells expressing SLUG shRNA to anticancer drugs. CONCLUSION We provide compelling evidence showing that PTEN is a direct functional target of SLUG. Our findings offer new insight in the regulation of the PTEN/AKT pathway and provide a molecular basis for potential targeted therapies of prostate cancer PMID:25728608

  9. 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. PMID:22551504

  10. 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.

  11. 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.

  12. 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. PMID:27270589

  13. 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

  14. The transcriptional repressor ICER binds to multiple loci throughout the genome.

    PubMed

    Muñiz, Luis C; Molina, Carlos A

    2016-09-23

    The events culminating in ovulation are controlled by the cyclical actions of hormones such as Follical Stimulating Hormone (FSH) and Luteinizing Hormone (LH). The secondary messenger, cyclic AMP (cAMP) conveys the intracellular activity of these hormones. It is well established that a family of transcription factors facilitate cAMP mediated gene expression, yet it remains unknown how these factors directly affect ovulation. One of these factors, Inducible cAMP Early Repressor (ICER) has been implicated in the transcriptional regulation of cAMP inducible genes during folliculogenesis and ovulation. In order to better determine the role of ICER in ovarian function we have identified novel targets using a genome-wide approach. Using a modification of the chromatin immunoprecipitation (ChIP) assay we directly cloned and sequenced the immunoprecipitated ICER-associated DNAs from an immortalized mouse granulose cell line (GRMO2). The analysis of the immunoprecipitated DNA fragments has revealed that ICER's binding to DNA has the following distribution; 16% within the promoter region, 31% within an intron, 14% were not within a gene, 6% were within 20 kb of a promoter and 3% were within the 3' end of genes.

  15. Myeloid Deletion of Nemo Causes Osteopetrosis in Mice Owing to Upregulation of Transcriptional Repressors

    PubMed Central

    Swarnkar, Gaurav; Shim, Kyuhwan; Nasir, Amjad M.; Seehra, Kuljeet; Chen, Hung-Po (Tim); Mbalaviele, Gabriel; Abu-Amer, Yousef

    2016-01-01

    The transcription factor NF-κB is central to numerous physiologic processes including bone development, and its activation is controlled by IKKγ (also called NEMO), the regulatory subunit of IKK complex. NEMO is X-linked, and mutations in this gene result in Incontinentia Pigmenti in human hemizygous females. In mice, global deficiency causes embryonic lethality. In addition, certain point mutations in the NEMO (IKBKG) human gene manifest skeletal defects implicating NEMO in the regulation of bone homeostasis. To specifically investigate such role, we conditionally deleted Nemo from osteoclast and myeloid progenitors. Morphometric, histologic, and molecular analyses demonstrate that myeloid NEMO deletion causes osteopetrosis in mice. Mechanistically, NEMO deficiency hampered activation of IKK complex in osteoclast precursors, causing arrest of osteoclastogenesis and apoptosis. Interestingly, inhibiting apoptosis by genetic ablation of TNFr1 significantly increased cell survival, but failed to rescue osteoclastogenesis or reverse osteopetrosis. Based on this observation, we analyzed the expression of different regulators of osteoclastogenesis and discovered that NEMO deletion leads to increased RBPJ expression, resulting in a decrease of Blimp1 expression. Consequently, expression of IRF8 and Bcl6 which are targets of Blimp1 and potent osteoclastogenic transcriptional repressors, is increased. Thus, NEMO governs survival and osteoclast differentiation programs through serial regulation of multiple transcription factors. PMID:27435916

  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. Distinct structural features of Rex-family repressors to sense redox levels in anaerobes and aerobes.

    PubMed

    Zheng, Yingying; Ko, Tzu-Ping; Sun, Hong; Huang, Chun-Hsiang; Pei, Jianjun; Qiu, Riyong; Wang, Andrew H-J; Wiegel, Juergen; Shao, Weilan; Guo, Rey-Ting

    2014-12-01

    The Rex-family repressors sense redox levels by alternative binding to NADH or NAD(+). Unlike other Rex proteins that regulate aerobic respiration, RSP controls ethanol fermentation in the obligate anaerobe Thermoanaerobacter ethanolicus JW200(T). It is also found in other anaerobic microorganisms. Here we present the crystal structures of apo-RSP, RSP/NADH and RSP/NAD(+)/DNA, which are the first structures of Rex-family members from an obligate anaerobe. RSP functions as a homodimer. It assumes an open conformation when bound to the operator DNA and a closed conformation when not DNA-bound. The DNA binds to the N-terminal winged-helix domain and the dinucleotide, either reduced or oxidized, binds to the C-terminal Rossmann-fold domain. The two distinct orientations of nicotinamide ring, anti in NADH and syn in NAD(+), give rise to two sets of protein-ligand interactions. Consequently, NADH binding makes RSP into a closed conformation, which does not bind to DNA. Both the conserved residues and the DNA specificity of RSP show a number of variations from those of the aerobic Rex, reflecting different structural bases for redox-sensing by the anaerobic and aerobic Rex-family members. PMID:25463021

  18. 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.

  19. 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.

  20. 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

  1. Allosteric inhibition of a zinc-sensing transcriptional repressor: Insights into the arsenic repressor (ArsR) family

    PubMed Central

    Campanello, Gregory C.; Ma, Zhen; Grossoehme, Nicholas E.; Guerra, Alfredo J.; Ward, Brian P.; DiMarchi, Richard D.; Ye, Yuzhen; Dann, Charles E.; Giedroc, David P.

    2013-01-01

    The molecular basis of allosteric regulation remains a subject of intense interest. Staphylococcus aureus CzrA is a member of the ubiquitous arsenic repressor (ArsR) family of bacterial homodimeric metal sensing proteins, and has emerged as a model system for understanding allosteric regulation of operator DNA binding by transition metal ions. Using unnatural amino acid substitution and a standard linkage analysis, we show that a His97’ NHε2•••O=C-His67 quaternary structural hydrogen bond is an energetically significant contributor to the magnitude of the allosteric coupling free energy, ΔGc. A “cavity” introduced just beneath this hydrogen bond in V66A/L68V CzrA results in a dramatic loss of regulation by Zn(II) despite adopting a wild-type global structure and Zn(II) binding and DNA binding affinities only minimally affected from wild-type. The energetics of Zn(II) binding and heterotropic coupling free energies (ΔHc, −TΔSc) of the double mutant are also radically altered and suggest that increased internal dynamics leads to poorer allosteric negative regulation in V66A/L68V CzrA. A statistical coupling analysis of 3000 ArsR proteins reveals a sector that links the DNA-binding determinants and the α5 Zn(II) sensing sites through V66/L68 in CzrA. We propose that distinct regulatory sites uniquely characteristic of individual ArsR proteins results from evolution of distinct connectivities to this sector, each capable of driving the same biological outcome, transcriptional derepression. PMID:23353829

  2. Comparing native and irradiated E. coli lactose repressor-operator complex by molecular dynamics simulation.

    PubMed

    Aci-Sèche, Samia; Garnier, Norbert; Goffinont, Stéphane; Genest, Daniel; Spotheim-Maurizot, Mélanie; Genest, Monique

    2010-09-01

    The function of the E. coli lactose operon requires the binding of the tetrameric repressor protein to the operator DNA. We have previously shown that gamma-irradiation destabilises the repressor-operator complex because the repressor gradually loses its DNA-binding ability (Radiat Res 170:604-612, 2008). It was suggested that the observed oxidation of tyrosine residues and the concomitant structural changes of irradiated headpieces (DNA-binding domains of repressor monomers) could be responsible for the inactivation. To unravel the mechanisms that lead to repressor-operator complex destabilisation when tyrosine oxidation occurs, we have compared by molecular dynamic simulations two complexes: (1) the native complex formed by two headpieces and the operator DNA, and (2) the damaged complex, in which all tyrosines are replaced by their oxidation product 3,4-dihydroxyphenylalanine (DOPA). On a 20 ns time scale, MD results show effects consistent with complex destabilisation: increased flexibility, increased DNA bending, modification of the hydrogen bond network, and decrease of the positive electrostatic potential at the protein surface and of the global energy of DNA-protein interactions. PMID:20349312

  3. 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.

  4. Interconvertible lac repressor-DNA loops revealed by single-molecule experiments.

    PubMed

    Wong, Oi Kwan; Guthold, Martin; Erie, Dorothy A; Gelles, Jeff

    2008-09-30

    At many promoters, transcription is regulated by simultaneous binding of a protein to multiple sites on DNA, but the structures and dynamics of such transcription factor-mediated DNA loops are poorly understood. We directly examined in vitro loop formation mediated by Escherichia coli lactose repressor using single-molecule structural and kinetics methods. Small ( approximately 150 bp) loops form quickly and stably, even with out-of-phase operator spacings. Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein-DNA complexes. The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic "V" conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain. The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions. PMID:18828671

  5. Site-specific DNA-affinity chromatography of the lac repressor.

    PubMed Central

    Herrick, G

    1980-01-01

    To test the feasibility of site-specific DNA-affinity chromatography, E. coli lac repressor was bound to an operator-containing DNA column, and in parallel to a non-operator DNA column. Salt gradient elution shows: 1) elution from non-operator DNA was near 250mM KCl or NaCl; interpretation of this result suggests the usefulness of the procedure for studying salt-dependence of DNA-protein affinities; 2) elution from operator-containing DNA was delayed (average elution = 1000mM salt), demonstrating a feasibility of site-specific DNA-affinity chromatography, if one provides a sufficiently favorable ratio of specific to non-specific DNA binding sites; 3) repressor eluted from operator-containing DNA over a very broad salt range, which may represent chromatography-generated repressor heterogeneity. PMID:7001362

  6. The trigger enzyme PepA (aminopeptidase A) of Escherichia coli, a transcriptional repressor that generates positive supercoiling.

    PubMed

    Nguyen Le Minh, Phu; Nadal, Marc; Charlier, Daniel

    2016-06-01

    Escherichia coli aminopeptidase A (PepA) is a trigger enzyme endowed with catalytic activity and DNA-binding properties prominent in transcriptional regulation and site-specific DNA recombination. The current work demonstrates that PepA is a repressor in its own right, capable of specifically inhibiting transcription initiation at promoter P1 of the carAB operon, encoding carbamoylphosphate synthase. Furthermore, in vitro topology studies performed with DNA minicircles demonstrate that PepA binding constrains a single positive supercoil in the carP1 control region. Such a topological event is understood to constitute an impediment to transcription initiation and may serve as a mechanism to regulate gene expression.

  7. Mouse Hobit is a homolog of the transcriptional repressor Blimp-1 that regulates NKT cell effector differentiation.

    PubMed

    van Gisbergen, Klaas P J M; Kragten, Natasja A M; Hertoghs, Kirsten M L; Wensveen, Felix M; Jonjic, Stipan; Hamann, Jörg; Nolte, Martijn A; van Lier, Rene A W

    2012-09-01

    The transcriptional repressor Blimp-1 mediates the terminal differentiation of many cell types, including T cells. Here we identified Hobit (Znf683) as a previously unrecognized homolog of Blimp-1 that was specifically expressed in mouse natural killer T cells (NKT cells). Through studies of Hobit-deficient mice, we found that Hobit was essential for the formation of mature thymic NKT cells. In the periphery, Hobit repressed the accumulation of interferon-γ (IFN-γ)-producing NK1.1(lo) NKT cells at steady state. After antigenic stimulation, Hobit repressed IFN-γ expression, whereas after innate stimulation, Hobit induced granzyme B expression. Thus, reminiscent of the function of Blimp-1 in other lymphocytes, Hobit controlled the maintenance of quiescent, fully differentiated NKT cells and regulated their immediate effector functions.

  8. Calorimetric analysis of lambda cI repressor binding to DNA operator sites.

    PubMed

    Merabet, E; Ackers, G K

    1995-07-11

    Enthalpies and heat capacities were determined by isothermal titration calorimetry for bacteriophage lambda cI repressor binding to DNA containing various combinations of the three operator sites OR1, OR2, and OR3 (each comprising a consensus half-site and a specific nonconsensus half-site). Differential scanning calorimetry was employed to evaluate the effects of specific DNA binding on thermal melting of the N-terminal and C-terminal repressor domains. Principal findings of this study are as follows: (1) Binding of repressor to each of the DNA operators is dominated by a large negative enthalpy, in agreement with earlier van't Hoff analyses of quantitative footprint titration data [Koblan & Ackers (1992) Biochemistry 31, 57-65]. The calorimetric data also revealed negative heat capacities for cI binding that are of comparable magnitude with many other systems [Spolar & Record (1994) Science 263, 777-784]. However, this feature in combination with the large negative values of binding enthalpies leads to an enthalpic dominance throughout the physiological temperature range. The resulting thermodynamic profile is opposite to the entropically dominated binding observed for many systems, including lambda cro repressor which binds to the same sites as cI and also employs a helix-turn-helix binding domain [Takeda et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8180-8184]. It is suggested that these thermodynamic differences may arise from interactions of the cI repressor's N-terminal "arm" with the DNA. (2) Repressor monomers do not bind significantly to DNA containing either a consensus half-site or a nonconsensus half-site. Binding affinity to the double-consensus operator is much weaker than to any of the natural full-site operators. The same was found with other combinations of half-sites. A mutant repressor (PT158) which is severely defective in dimerization [Burz et al. (1994) Biochemistry 33, 8399-8405] was also found to bind only full-site operators and showed

  9. DNA sequence dependent and independent conformational changes in multipartite operator recognition by lambda-repressor.

    PubMed

    Deb, S; Bandyopadhyay, S; Roy, S

    2000-03-28

    Binding of regulatory proteins to multipartite DNA binding sites often occurs with protein-protein interaction, resulting in cooperative binding. The operators of bacteriophage lambda have several pairs of repressor binding sites (O(R)1-O(R)2, O(R)2-O(R)3, O(L)1-O(L)2, and O(L)2-O(L)3) separated by a variable number of base pairs, and thus, bacteriophage lambda is a model system for studying multipartite operator recognition by DNA-binding proteins. Near-UV circular dichroism spectra show that the DNA is distorted in O(R)1-O(R)2 and O(L)2-O(L)3 but much less so in O(R)2-O(R)3. Upon titration of lambda-repressor with single-operator sites O(R)1, O(R)2, and O(R)3, it was observed that the tryptophan fluorescence quenches to different degrees, suggesting different conformations of the protein in the three DNA-protein complexes. Acrylamide quenching of tryptophan fluorescence of lambda-repressor bound to these single operators also shows different Stern-Volmer constants, supporting the above conclusions. Titration of lambda-repressor with oligonucleotides containing pairs of operator sites also causes different degrees of fluorescence quenching. In particular, fluorescence quenching induced by O(R)1-O(R)2 binding is less than the quenching induced by either of the single operators alone, suggesting additional conformational changes upon establishment of protein-protein contact. Stern-Volmer constants obtained from acrylamide quenching of tryptophan fluorescence of lambda-repressor bound cooperatively to pairs of operator sites are different from those of the single-operator-site-bound repressors. For example, O(R)2-O(R)3-bound repressor has significantly higher acrylamide quenchable components than either of the O(R)2- or O(R)3-bound proteins, again suggesting additional conformational changes upon establishment of protein-protein contact. We conclude that the strategy of recognition of multipartite operator by lambda-repressor is complex and varied, involving

  10. 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

  11. Regulation of Pyrimidine Biosynthetic Gene Expression in Bacteria: Repression without Repressors

    PubMed Central

    Turnbough, Charles L.; Switzer, Robert L.

    2008-01-01

    Summary: DNA-binding repressor proteins that govern transcription initiation in response to end products generally regulate bacterial biosynthetic genes, but this is rarely true for the pyrimidine biosynthetic (pyr) genes. Instead, bacterial pyr gene regulation generally involves mechanisms that rely only on regulatory sequences embedded in the leader region of the operon, which cause premature transcription termination or translation inhibition in response to nucleotide signals. Studies with Escherichia coli and Bacillus subtilis pyr genes reveal a variety of regulatory mechanisms. Transcription attenuation via UTP-sensitive coupled transcription and translation regulates expression of the pyrBI and pyrE operons in enteric bacteria, whereas nucleotide effects on binding of the PyrR protein to pyr mRNA attenuation sites control pyr operon expression in most gram-positive bacteria. Nucleotide-sensitive reiterative transcription underlies regulation of other pyr genes. With the E. coli pyrBI, carAB, codBA, and upp-uraA operons, UTP-sensitive reiterative transcription within the initially transcribed region (ITR) leads to nonproductive transcription initiation. CTP-sensitive reiterative transcription in the pyrG ITRs of gram-positive bacteria, which involves the addition of G residues, results in the formation of an antiterminator RNA hairpin and suppression of transcription attenuation. Some mechanisms involve regulation of translation rather than transcription. Expression of the pyrC and pyrD operons of enteric bacteria is controlled by nucleotide-sensitive transcription start switching that produces transcripts with different potentials for translation. In Mycobacterium smegmatis and other bacteria, PyrR modulates translation of pyr genes by binding to their ribosome binding site. Evidence supporting these conclusions, generalizations for other bacteria, and prospects for future research are presented. PMID:18535147

  12. Superinduction of metallothionein I by inhibition of protein synthesis: role of a labile repressor in MTF-1 mediated gene transcription.

    PubMed

    Bi, Yongyi; Lin, Gary X; Millecchia, Lyndell; Ma, Qiang

    2006-01-01

    Induction of metallothioneins (MTs) through the metal-activated transcription factor-1 (MTF-1) provides a model response for analyzing transcriptional gene regulation by heavy metals. Here, we report inhibition of protein synthesis by cycloheximide (CHX) increases induction of Mt1 by approximately five-fold, a phenomenon designated as "superinduction." Characterization of superinduction revealed it is time- and concentration-dependent of CHX, requires the presence of an MTF-1 activator, and occurs at a transcriptional level, suggesting a labile repressor in the control of Mt1 induction. Genetic analyses using Mtf1 null cells and a metal response element (MRE)-driven reporter construct showed that superinduction of Mt1 is mediated through MTF-1 and MRE-dependent transcription. Analyses of intracellular zinc content by inductively coupled plasma emission spectroscopy and fluorescence imaging demonstrated that treatment with CHX alone or CHX plus an inducer does not increase the total zinc accumulation or the concentration of free zinc in cells under the conditions in which superinduction occurs. Moreover, superinduction was observed in cells cultured in a zinc-depleted medium, suggesting that superinduction does not involve elevation of intracellular zinc concentration. Northern blotting showed that Cd, CHX, or Cd + CHX does not affect the expression of the mRNA of MTF-1. Immunoblotting using antibodies specific for MTF-1 demonstrated that Cd induces a down-regulation of the MTF-1 protein, whereas cotreatment with Cd and CHX blocked the Cd-induced degradation of MTF-1. The findings reveal a new mechanistic aspect of the superinduction of Mt1, in which a labile repressor negatively controls agonist-induced turnover of the MTF-1 protein. PMID:16615093

  13. Evidence-Based Structural Model of the Staphylococcal Repressor Protein: Separation of Functions into Different Domains

    PubMed Central

    Nyíri, Kinga; Kőhegyi, Bianka; Micsonai, András; Kardos, József; Vertessy, Beata G.

    2015-01-01

    Horizontal transfer of mobile genetic elements within Staphylococci is of high biomedical significance as such elements are frequently responsible for virulence and toxic effects. Staphylococcus-encoded repressor proteins regulate the replication of these mobile genetic elements that are located within the so-called pathogenicity islands. Here, we report structural and functional characterization of one such repressor protein, namely the Stl protein encoded by the pathogenicity island SaPIbov1. We create a 3D structural model and based on this prediction, we investigate the different functionalities of truncated and point mutant constructs. Results suggest that a helix-turn-helix motif governs the interaction of the Stl protein with its cognate DNA site: point mutations within this motif drastically decrease DNA-binding ability, whereas the interaction with the Stl-binding partner protein dUTPase is unperturbed by these point mutations. The 3D model also suggested the potential independent folding of a carboxy-terminal domain. This suggestion was fully verified by independent experiments revealing that the carboxy-terminal domain does not bind to DNA but is still capable of binding to and inhibiting dUTPase. A general model is proposed, which suggests that among the several structurally different repressor superfamilies Stl-like Staphylococcal repressor proteins belong to the helix-turn-helix transcription factor group and the HTH motif is suggested to reside within N-terminal segment. PMID:26414067

  14. Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor.

    PubMed

    Gillard, Nathalie; Goffinont, Stephane; Buré, Corinne; Davidkova, Marie; Maurizot, Jean-Claude; Cadene, Martine; Spotheim-Maurizot, Melanie

    2007-05-01

    Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece. PMID:17263689

  15. In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli

    PubMed Central

    2014-01-01

    A multitude of studies have looked at the in vivo and in vitro behavior of the lac repressor binding to DNA and effector molecules in order to study transcriptional repression, however these studies are not always reconcilable. Here we use in vitro transcription to directly mimic the in vivo system in order to build a self consistent set of experiments to directly compare in vivo and in vitro genetic repression. A thermodynamic model of the lac repressor binding to operator DNA and effector is used to link DNA occupancy to either normalized in vitro mRNA product or normalized in vivo fluorescence of a regulated gene, YFP. An accurate measurement of repressor, DNA and effector concentrations were made both in vivo and in vitro allowing for direct modeling of the entire thermodynamic equilibrium. In vivo repression profiles are accurately predicted from the given in vitro parameters when molecular crowding is considered. Interestingly, our measured repressor–operator DNA affinity differs significantly from previous in vitro measurements. The literature values are unable to replicate in vivo binding data. We therefore conclude that the repressor-DNA affinity is much weaker than previously thought. This finding would suggest that in vitro techniques that are specifically designed to mimic the in vivo process may be necessary to replicate the native system. PMID:25097824

  16. Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

    PubMed Central

    Gillard, Nathalie; Goffinont, Stephane; Buré, Corinne; Davidkova, Marie; Maurizot, Jean-Claude; Cadene, Martine; Spotheim-Maurizot, Melanie

    2007-01-01

    Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with γ-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH· radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH· radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece. PMID:17263689

  17. Dynamical behavior of the activator-repressor circuit model under random fluctuations

    NASA Astrophysics Data System (ADS)

    Lee, Jae Kwan; Cinar, Ali; Duan, Jinqiao

    2011-04-01

    The purpose of this paper is to investigate possible impact of uncertainty on dynamical evolution of an activator-repressor circuit model, for some gene regulatory networks. Escape probability and mean residence time are computed in order to gain insights on the role played by random fluctuations. Some changes or bifurcations in mean residence time are also observed when key model parameters vary.

  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. hnRNP I/PTB can antagonize the splicing repressor activity of SRp30c

    PubMed Central

    Paradis, Caroline; Cloutier, Philippe; Shkreta, Lulzim; Toutant, Johanne; Klarskov, Klaus; Chabot, Benoit

    2007-01-01

    The control of alternative pre-mRNA splicing often requires the participation of factors displaying synergistic or antagonistic activities. In the hnRNP A1 pre-mRNA, three elements promote the exclusion of alternative exon 7B, while a fourth intron element (CE9) represses splicing of exon 7B to the downstream exon. We have shown previously that the 5′ portion of the 38-nucleotide-long CE9 element is bound by SRp30c, and that this interaction is important for repression in vitro. To determine whether SRp30c alone can impose repression, we tested a high-affinity SRp30c binding site that we identified using the SELEX protocol. We find that multiple high-affinity SRp30c sites are required to replicate the level of repression obtained with CE9, and that both the 5′ and the 3′ portions of CE9 contribute to SRp30c binding. Performing RNA affinity chromatography with the complete CE9 element recovered hnRNP I/PTB. Surprisingly however, His-tagged PTB reduced the binding of SRp30c to CE9 in a nuclear extract, stimulated splicing to a downstream 3′ splice site, and relieved the CE9-mediated splicing repression in vitro. Our in vivo results are consistent with the notion that increasing PTB levels alleviates the repression imposed by CE9 to a downstream 3′ splice site. Thus, PTB can function as an anti-repressor molecule to counteract the splicing inhibitory activity of SRp30c. PMID:17548433

  20. 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

  1. 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

  2. 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

  3. 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.

  4. The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation.

    PubMed

    Dill, Alyssa; Thomas, Stephen G; Hu, Jianhong; Steber, Camille M; Sun, Tai-Ping

    2004-06-01

    The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box-containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCFSLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCFSLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein-protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins. PMID:15155881

  5. 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

  6. Association of micropenis with Pro185Ala polymorphism of the gene for aryl hydrocarbon receptor repressor involved in dioxin signaling.

    PubMed

    Soneda, Shun; Fukami, Maki; Fujimoto, Masatoshi; Hasegawa, Tomonobu; Koitabashi, Yasushi; Ogata, Tsutomu

    2005-02-01

    The prevalence of undermasculinized external genitalia has increased in several countries including Japan, and this phenomenon has primarily been ascribed to the deleterious effects of environmental endocrine disruptors such as dioxins. To examine a possible role of the genetic susceptibility to dioxins in the development of micropenis (MP), we studied the Arg554Lys polymorphism of the gene for aryl hydrocarbon receptor (AHR) and the Pro185Ala polymorphism of the gene for aryl hydrocarbon receptor repressor (AHRR), in 73 boys with MP (34 boys with mild MP from -2.1 to -2.5 SD and 39 boys with severe MP below -2.5 SD) and 80 control males (50 boys and 30 fertile adult males). The allele and genotype frequencies of the AHR polymorphism were comparable between the two groups of males, but those of the AHRR polymorphism were significantly different, with the Pro allele and the Pro/Pro genotype being more frequent in boys with MP than in control males (P-value: 0.0029 for the allele frequency and 0.011 for the genotype frequency). In addition, both polymorphisms were comparable in the allele and genotype frequencies between boys with mild MP and those with severe MP and between control boys and control fertile adult males. The results suggest that the AHRR Pro185Ala polymorphism may constitute a susceptibility locus for the development of MP in response to dioxins.

  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. ARA67/PAT1 Functions as a Repressor To Suppress Androgen Receptor Transactivation

    PubMed Central

    Zhang, Yanqing; Yang, Yue; Yeh, Shuyuan; Chang, Chawnshang

    2004-01-01

    The androgen receptor (AR) may recruit multiple coregulators for proper or optimal transactivation. Here we report the identification and characterization of ARA67/PAT1 as an AR coregulator from a prostate cDNA library. ARA67/PAT1 was screened out as an AR N terminus interacting protein. Interaction mapping shows that the cooperation of multiple domains within ARA67/PAT1 may be required for the maximal interaction with AR. ARA67/PAT1 functions as a repressor with better suppressive effects on AR compared to glucocorticoid receptor and estrogen receptor. Further mechanism dissection reveals that the interrupted AR cytoplasmic-nuclear shuttling may play a major role in ARA67/PAT1 mediated suppression on AR. Together, these results suggest that ARA67/PAT1 may function as a novel repressor that can modulate AR function in prostate cancer. PMID:14729952

  9. The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor.

    PubMed

    Huang, M; Zhou, Z; Elledge, S J

    1998-09-01

    We have identified the yeast CRT1 gene as an effector of the DNA damage and replication checkpoint pathway. CRT1 encodes a DNA-binding protein that recruits the general repressors Ssn6 and Tup1 to the promoters of damage-inducible genes. Derepression of the Crt1 regulon suppresses the lethality of mec1 and rad53 null alleles and is essential for cell viability during replicative stress. In response to DNA damage and replication blocks, Crt1 becomes hyperphosphorylated and no longer binds DNA, resulting in transcriptional induction. CRT1 is autoregulated and is itself induced by DNA damage, indicating the existence of a negative feedback pathway that facilitates return to the repressed state after elimination of damage. The inhibition of an autoregulatory repressor in response to DNA damage is a strategy conserved throughout prokaryotic and eukaryotic evolution.

  10. 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.

  11. Arabidopsis transcriptional repressor VAL1 triggers Polycomb silencing at FLC during vernalization.

    PubMed

    Qüesta, Julia I; Song, Jie; Geraldo, Nuno; An, Hailong; Dean, Caroline

    2016-07-29

    The determinants that specify the genomic targets of Polycomb silencing complexes are still unclear. Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC) accelerates flowering and involves a cold-dependent epigenetic switch. Here we identify a single point mutation at an intragenic nucleation site within FLC that prevents this epigenetic switch from taking place. The mutation blocks nucleation of plant homeodomain-Polycomb repressive complex 2 (PHD-PRC2) and indicates a role for the transcriptional repressor VAL1 in the silencing mechanism. VAL1 localizes to the nucleation region in vivo, promoting histone deacetylation and FLC transcriptional silencing, and interacts with components of the conserved apoptosis- and splicing-associated protein (ASAP) complex. Sequence-specific targeting of transcriptional repressors thus recruits the machinery for PHD-PRC2 nucleation and epigenetic silencing.

  12. 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

  13. cAMP inducibility of transcriptional repressor ICER in developing and mature human T lymphocytes.

    PubMed

    Bodor, J; Spetz, A L; Strominger, J L; Habener, J F

    1996-04-16

    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.

  14. [Spatial structure of cro-repressor in a solution. II. Effect of ionized groups].

    PubMed

    Kurochkin, A V; Kirpichnikov, M P

    1986-01-01

    The technique of 1H NMR spectroscopy and absorption UV spectroscopy were used to study the ionization of the tyrosine phenol cycles and the effect of ionizable groups on the chemical shifts of signals from protons in the side chains of several amino acid residues. The microenvironment of these residues was established by analysing the titration curves. The mutual orientation of two functionally important adjacent alpha-helical protein regions was determined in solution. The signals from methionine residues belonging to different regions of the secondary structure were assigned in the NMR spectrum. The results indicate that the spatial structure of the repressor is similar in solution an in the crystal. They confirm the model proposed for the cro repressor interaction with DNA and based on the data of X-ray diffraction analysis.

  15. Purification and characterization of nucleolin and its identification as a transcription repressor.

    PubMed Central

    Yang, T H; Tsai, W H; Lee, Y M; Lei, H Y; Lai, M Y; Chen, D S; Yeh, N H; Lee, S C

    1994-01-01

    Expression of the acute-phase response genes, such as that for alpha-1 acid glycoprotein (AGP), involves both positive and negative transcription factors. A positive transcription factor, AGP/EBP, and a negative transcription factor, factor B, have been identified as the two most important factors responsible for the induction of the AGP gene. In this paper we report the purification, characterization, and identification of a B-motif-binding factor from the mouse hepatoma cell line 129p. The purified factor has been identified as nucleolin by amino acid sequence analysis. Biochemical and functional studies further established that nucleolin is a transcription repressor for regulation of AGP and possibly other acute-phase response genes. Thus, in addition to the many known functions of nucleolin, such as rRNA transcription, processing, ribosome biogenesis, and the shuttling of proteins between the cytoplasmic and nuclear compartments, it may also function as a transcriptional repressor. Images PMID:8065340

  16. Arabidopsis transcriptional repressor VAL1 triggers Polycomb silencing at FLC during vernalization.

    PubMed

    Qüesta, Julia I; Song, Jie; Geraldo, Nuno; An, Hailong; Dean, Caroline

    2016-07-29

    The determinants that specify the genomic targets of Polycomb silencing complexes are still unclear. Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC) accelerates flowering and involves a cold-dependent epigenetic switch. Here we identify a single point mutation at an intragenic nucleation site within FLC that prevents this epigenetic switch from taking place. The mutation blocks nucleation of plant homeodomain-Polycomb repressive complex 2 (PHD-PRC2) and indicates a role for the transcriptional repressor VAL1 in the silencing mechanism. VAL1 localizes to the nucleation region in vivo, promoting histone deacetylation and FLC transcriptional silencing, and interacts with components of the conserved apoptosis- and splicing-associated protein (ASAP) complex. Sequence-specific targeting of transcriptional repressors thus recruits the machinery for PHD-PRC2 nucleation and epigenetic silencing. PMID:27471304

  17. Ethylene regulates arabidopsis development via the modulation of DELLA protein growth repressor function.

    PubMed

    Achard, Patrick; Vriezen, Wim H; Van Der Straeten, Dominique; Harberd, Nicholas P

    2003-12-01

    Phytohormones regulate plant development via a poorly understood signal response network. Here, we show that the phytohormone ethylene regulates plant development at least in part via alteration of the properties of DELLA protein nuclear growth repressors, a family of proteins first identified as gibberellin (GA) signaling components. This conclusion is based on the following experimental observations. First, ethylene inhibited Arabidopsis root growth in a DELLA-dependent manner. Second, ethylene delayed the GA-induced disappearance of the DELLA protein repressor of ga1-3 from root cell nuclei via a constitutive triple response-dependent signaling pathway. Third, the ethylene-promoted "apical hook" structure of etiolated seedling hypocotyls was dependent on the relief of DELLA-mediated growth restraint. Ethylene, auxin, and GA responses now can be attributed to effects on DELLA function, suggesting that DELLA plays a key integrative role in the phytohormone signal response network.

  18. LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain

    PubMed Central

    Wang, Yixuan; Wu, Qian; Yang, Peng; Wang, Chenfei; Liu, Jing; Ding, Wenyu; Liu, Wensu; Bai, Ye; Yang, Yuanyuan; Wang, Hong; Gao, Shaorong; Wang, Xiaoqun

    2016-01-01

    Epigenetic regulatory complexes play key roles in the modulation of transcriptional regulation underlying neural stem cell (NSC) proliferation and progeny specification. How specific cofactors guide histone demethylase LSD1/KDM1A complex to regulate distinct NSC-related gene activation and repression in cortical neurogenesis remains unclear. Here we demonstrate that Rcor2, a co-repressor of LSD1, is mainly expressed in the central nervous system (CNS) and plays a key role in epigenetic regulation of cortical development. Depletion of Rcor2 results in reduced NPC proliferation, neuron population, neocortex thickness and brain size. We find that Rcor2 directly targets Dlx2 and Shh, and represses their expressions in developing neocortex. In addition, inhibition of Shh signals rescues the neurogenesis defects caused by Rcor2 depletion both in vivo and in vitro. Hence, our findings suggest that co-repressor Rcor2 is critical for cortical development by repressing Shh signalling pathway in dorsal telencephalon. PMID:26795843

  19. A System of Repressor Gradients Spatially Organizes the Boundaries of “Morphogen-dependent” Target Genes

    PubMed Central

    Chen, Hongtao; Xu, Zhe; Mei, Constance; Yu, Danyang; Small, Stephen

    2012-01-01

    Summary The homeodomain (HD) protein Bicoid (Bcd) is thought to function as a gradient morphogen that positions boundaries of target genes via threshold-dependent activation mechanisms. Here we analyze 66 Bcd-dependent regulatory elements, and show that their boundaries are positioned primarily by repressive gradients that antagonize Bcd-mediated activation. A major repressor is the pair-rule protein Runt, which is expressed in an opposing gradient, and is necessary and sufficient for limiting Bcd-dependent activation. Evidence is presented that Runt functions with the maternal repressor Capicua and the gap protein Kruppel as the principal components of a repression system that correctly orders boundaries throughout the anterior half of the embryo. These results put conceptual limits on the Bcd morphogen hypothesis, and demonstrate how the Bcd gradient functions within the gene network that patterns the embryo. PMID:22541432

  20. 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.

  1. Energy transfer in lactose repressor protein modified with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate.

    PubMed

    Gardner, J A; Matthews, K S

    1991-03-12

    Energy transfer between the two tryptophan residues in the lactose repressor protein and the fluorescent moiety of the cysteine-specific reagent N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonate (1,5-IAEDANS) has been examined. Modification of repressor with this compound did not affect operator or inducer binding. 1,5-IAEDANS reacted primarily with Cys140 in wild-type repressor [Schneider et al. (1984) Biochemistry 23, 2221]; in the presence of inducer, modification at Cys107 increased, while reaction at Cys140 remained unchanged. Energy transfer between tryptophans and the AEDANS moiety(ies) in wild-type lac repressor occurred with an efficiency of 6.7 +/- 1.9% in the absence and 7.8 +/- 1.6% in the presence of inducer. The distance between the Trp donor(s) and the acceptor in wild-type repressor was calculated to be in the range approximately 35 A under both conditions. The similarity in efficiency despite large differences in the amount of acceptor attached to Cys107 when inducer is bound indicates that the AEDANS group at position 107 does not participate significantly in energy transfer and that the label at position 140 acts as the primary acceptor group. The similarity of energy-transfer efficiency (7.1 +/- 3.8%) observed for 1,5-IAEDANS-modified monomeric mutant repressor (Y282D) indicates that the transfer is primarily intrasubunit in the native tetramer. Measurements using two mutant repressors (each with a single tryptophan and modified with 1,5-IAEDANS) demonstrated that both tryptophans can serve as donor in the energy-transfer process. The W201Y repressor (containing Trp220) exhibited a transfer efficiency lower than wild type (5.6 +/- 2.4%), corresponding to a slightly larger distance between the donor-acceptor pair in this mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Cleavage of a putative metal permease in Chlamydia trachomatis yields an iron-dependent transcriptional repressor.

    PubMed

    Thompson, Christopher C; Nicod, Sophie S; Malcolm, Denise S; Grieshaber, Scott S; Carabeo, Rey A

    2012-06-26

    The regulation of iron homeostasis is essential for most organisms, because iron is required for a variety of conserved biochemical processes, yet can be toxic at high concentrations. Upon experiencing iron starvation in vitro, the obligate intracellular human pathogen Chlamydia trachomatis exhibits elevated expression of a putative iron-transport system encoded by the ytg operon. The third component of the ytg operon, CT069 (YtgCR), encodes a protein with two distinct domains: a membrane-anchored metal ion permease and a diphtheria toxin repressor (DtxR)-like transcriptional repressor. In this report, we demonstrate that the C-terminal domain of CT069 (YtgR) serves as an iron-dependent autorepressor of the ytg operon. Moreover, the nascent full-length metal permease-transcriptional repressor protein was processed during the course of infection, and heterologously when expressed in Escherichia coli. The products produced by heterologous cleavage in E. coli were functional in the repression of a reporter gene downstream of a putative YtgR operator. We report a bona fide mechanism of iron-dependent regulation of transcription in Chlamydia. Moreover, the unusual membrane permease-DNA-binding polypeptide fusion configuration was found in several bacteria. Therefore, the DNA-binding capability and liberation of the YtgR domain from a membrane-anchored permease in C. trachomatis could represent a previously uncharacterized mechanism for prokaryotic regulation of iron-homeostasis.

  3. The chromatin remodeling factor CHD5 is a transcriptional repressor of WEE1.

    PubMed

    Quan, Jinhua; Adelmant, Guillaume; Marto, Jarrod A; Look, A Thomas; Yusufzai, Timur

    2014-01-01

    Loss of the chromatin remodeling ATPase CHD5 has been linked to the progression of neuroblastoma tumors, yet the underlying mechanisms behind the tumor suppressor role of CHD5 are unknown. In this study, we purified the human CHD5 complex and found that CHD5 is a component of the full NuRD transcriptional repressor complex, which also contains methyl-CpG binding proteins and histone deacetylases. The CHD5/NuRD complex appears mutually exclusive with the related CHD4/NuRD complex as overexpression of CHD5 results in loss of the CHD4 protein in cells. Following a search for genes that are regulated by CHD5 in neuroblastoma cells, we found that CHD5 binds to and represses the G2/M checkpoint gene WEE1. Reintroduction of CHD5 into neuroblastoma cells represses WEE1 expression, demonstrating that CHD5 can function as a repressor in cells. A catalytically inactive mutant version of CHD5 is able to associate with a NuRD cofactor but fails to repress transcription. Our study shows that CHD5 is a NuRD-associated transcriptional repressor and identifies WEE1 as one of the CHD5-regulated genes that may link CHD5 to tumor suppression.

  4. Cu(I)-mediated Allosteric Switching in a Copper-sensing Operon Repressor (CsoR)*

    PubMed Central

    Chang, Feng-Ming James; Coyne, H. Jerome; Cubillas, Ciro; Vinuesa, Pablo; Fang, Xianyang; Ma, Zhen; Ma, Dejian; Helmann, John D.; García-de los Santos, Alejandro; Wang, Yun-Xing; Dann, Charles E.; Giedroc, David P.

    2014-01-01

    The copper-sensing operon repressor (CsoR) is representative of a major Cu(I)-sensing family of bacterial metalloregulatory proteins that has evolved to prevent cytoplasmic copper toxicity. It is unknown how Cu(I) binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes. A phylogenetic analysis of 227 DUF156 protein members, including biochemically or structurally characterized CsoR/RcnR repressors, reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)-bound Gt CsoR reveals that Cu(I) binding induces a kink in the α2-helix between two conserved copper-ligating residues and folds an N-terminal tail (residues 12–19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo-state with these dynamics quenched upon Cu(I) binding. Small angle x-ray scattering experiments on an N-terminally truncated Gt CsoR (Δ2–10) reveal that the Cu(I)-bound tetramer is hydrodynamically more compact than is the apo-state. The implications of these findings for the allosteric mechanisms of other CsoR/RcnR repressors are discussed. PMID:24831014

  5. 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.

  6. Repressing a repressor: gibberellin-induced rapid reduction of the RGA protein in Arabidopsis.

    PubMed

    Silverstone, A L; Jung, H S; Dill, A; Kawaide, H; Kamiya, Y; Sun, T P

    2001-07-01

    RGA (for repressor of ga1-3) and SPINDLY (SPY) are likely repressors of gibberellin (GA) signaling in Arabidopsis because the recessive rga and spy mutations partially suppressed the phenotype of the GA-deficient mutant ga1-3. We found that neither rga nor spy altered the GA levels in the wild-type or the ga1-3 background. However, expression of the GA biosynthetic gene GA4 was reduced 26% by the rga mutation, suggesting that partial derepression of the GA response pathway by rga resulted in the feedback inhibition of GA4 expression. The green fluorescent protein (GFP)-RGA fusion protein was localized to nuclei in transgenic Arabidopsis. This result supports the predicted function of RGA as a transcriptional regulator based on sequence analysis. Confocal microscopy and immunoblot analyses demonstrated that the levels of both the GFP-RGA fusion protein and endogenous RGA were reduced rapidly by GA treatment. Therefore, the GA signal appears to derepress the GA signaling pathway by degrading the repressor protein RGA. The effect of rga on GA4 gene expression and the effect of GA on RGA protein level allow us to identify part of the mechanism by which GA homeostasis is achieved.

  7. Accurate genetic switch in Escherichia coli: novel mechanism of regulation by co-repressor.

    PubMed

    Tabaka, Marcin; Cybulski, Olgierd; Hołyst, Robert

    2008-04-01

    Understanding a biological module involves recognition of its structure and the dynamics of its principal components. In this report we present an analysis of the dynamics of the repression module within the regulation of the trp operon in Escherichia coli. We combine biochemical data for reaction rate constants for the trp repressor binding to trp operator and in vivo data of a number of tryptophan repressors (TrpRs) that bind to the operator. The model of repression presented in this report greatly differs from previous mathematical models. One, two or three TrpRs can bind to the operator and repress the transcription. Moreover, reaction rates for detachment of TrpRs from the operator strongly depend on tryptophan (Trp) concentration, since Trp can also bind to the repressor-operator complex and stabilize it. From the mathematical modeling and analysis of reaction rates and equilibrium constants emerges a high-quality, accurate and effective module of trp repression. This genetic switch responds accurately to fast consumption of Trp from the interior of a cell. It switches with minimal dispersion when the concentration of Trp drops below a thousand molecules per cell. PMID:18313075

  8. Contextual interactions determine whether the Drosophila homeodomain protein, Vnd, acts as a repressor or activator

    PubMed Central

    Yu, Zhongxin; Syu, Li-Jyun; Mellerick, Dervla M.

    2005-01-01

    At the molecular level, members of the NKx2.2 family of transcription factors establish neural compartment boundaries by repressing the expression of homeobox genes specific for adjacent domains [Muhr et al. (2001) Cell, 104, 861–873; Weiss et al. (1998) Genes Dev., 12, 3591–3602]. The Drosophila homologue, vnd, interacts genetically with the high-mobility group protein, Dichaete, in a manner suggesting co-operative activation [Zhao and Skeath (2002) Development, 129, 1165–1174]. However, evidence for direct interactions and transcriptional activation is lacking. Here, we present molecular evidence for the interaction of Vnd and Dichaete that leads to the activation of target gene expression. Two-hybrid interaction assays indicate that Dichaete binds the Vnd homeodomain, and additional Vnd sequences stabilize this interaction. In addition, Vnd has two activation domains that are typically masked in the intact protein. Whether vnd can activate or repress transcription is context-dependent. Full-length Vnd, when expressed as a Gal4 fusion protein, acts as a repressor containing multiple repression domains. A divergent domain in the N-terminus, not found in vertebrate Vnd-like proteins, causes the strongest repression. The co-repressor, Groucho, enhances Vnd repression, and these two proteins physically interact. The data presented indicate that the activation and repression domains of Vnd are complex, and whether Vnd functions as a transcriptional repressor or activator depends on both intra- and inter-molecular interactions. PMID:15640442

  9. The Chromatin Remodeling Factor CHD5 Is a Transcriptional Repressor of WEE1

    PubMed Central

    Quan, Jinhua; Adelmant, Guillaume; Marto, Jarrod A.; Look, A. Thomas; Yusufzai, Timur

    2014-01-01

    Loss of the chromatin remodeling ATPase CHD5 has been linked to the progression of neuroblastoma tumors, yet the underlying mechanisms behind the tumor suppressor role of CHD5 are unknown. In this study, we purified the human CHD5 complex and found that CHD5 is a component of the full NuRD transcriptional repressor complex, which also contains methyl-CpG binding proteins and histone deacetylases. The CHD5/NuRD complex appears mutually exclusive with the related CHD4/NuRD complex as overexpression of CHD5 results in loss of the CHD4 protein in cells. Following a search for genes that are regulated by CHD5 in neuroblastoma cells, we found that CHD5 binds to and represses the G2/M checkpoint gene WEE1. Reintroduction of CHD5 into neuroblastoma cells represses WEE1 expression, demonstrating that CHD5 can function as a repressor in cells. A catalytically inactive mutant version of CHD5 is able to associate with a NuRD cofactor but fails to repress transcription. Our study shows that CHD5 is a NuRD-associated transcriptional repressor and identifies WEE1 as one of the CHD5-regulated genes that may link CHD5 to tumor suppression. PMID:25247294

  10. 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.

  11. Escherichia coli gene purR encoding a repressor protein for purine nucleotide synthesis. Cloning, nucleotide sequence, and interaction with the purF operator.

    PubMed

    Rolfes, R J; Zalkin, H

    1988-12-25

    The Escherichia coli gene purR, encoding a repressor protein, was cloned by complementation of a purR mutation. Gene purR on a multicopy plasmid repressed expression of purF and purF-lacZ and reduced the growth rate of host cells by limiting the rate of de novo purine nucleotide synthesis. The level of a 1.3-kilobase purR mRNA was higher in cells grown with excess adenine, suggesting that synthesis of the repressor may be regulated. The chromosomal locus of purR was mapped to coordinate 1755-kb on the E. coli restriction map (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508). Pur repressor bound specifically to purF operator DNA as determined by gel retardation and DNase I footprinting assays. The amino acid sequence of Pur repressor was derived from the nucleotide sequence. Pur repressor subunit contains 341 amino acids and has a calculated Mr of 38,179. Pur repressor is 31-35% identical with the galR and cytR repressors and 26% identical with the lacI repressor. These four repressors are likely homologous. Amino acid sequence similarity is greatest in an amino-terminal region presumed to contain a DNA-binding domain. A similarity is also noted in the operator sites for these repressors.

  12. 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. PMID:21328406

  13. A new binding motif for the transcriptional repressor REST uncovers large gene networks devoted to neuronal functions.

    PubMed

    Otto, Stefanie J; McCorkle, Sean R; Hover, John; Conaco, Cecilia; Han, Jong-Jin; Impey, Soren; Yochum, Gregory S; Dunn, John J; Goodman, Richard H; Mandel, Gail

    2007-06-20

    The repressor element 1 (RE1) silencing transcription factor (REST) helps preserve the identity of nervous tissue by silencing neuronal genes in non-neural tissues. Moreover, in an epithelial model of tumorigenesis, loss of REST function is associated with loss of adhesion, suggesting the aberrant expression of REST-controlled genes encoding this property. To date, no adhesion molecules under REST control have been identified. Here, we used serial analysis of chromatin occupancy to perform genome-wide identification of REST-occupied target sequences (RE1 sites) in a kidney cell line. We discovered novel REST-binding motifs and found that the number of RE1 sites far exceeded previous estimates. A large family of targets encoding adhesion proteins was identified, as were genes encoding signature proteins of neuroendocrine tumors. Unexpectedly, genes considered exclusively non-neuronal also contained an RE1 motif and were expressed in neurons. This supports the model that REST binding is a critical determinant of neuronal phenotype.

  14. Multifunctional repressor KorB can block transcription by preventing isomerization of RNA polymerase-promoter complexes.

    PubMed Central

    Williams, D R; Motallebi-Veshareh, M; Thomas, C M

    1993-01-01

    The KorB protein of broad-host-range plasmid RK2 is a transcriptional repressor involved in the control of genes for plasmid replication, conjugative transfer and stable maintenance. We have purified this protein close to homogeneity from cells harbouring an overexpression vector with the korB gene under the control of the tac promoter. KorB binds to restriction fragments bearing its proposed operator sequence, OB. Its interaction with this palindromic site was confirmed by DNaseI or hydroxyl radical footprinting at two OB sequences from RK2. Comparisons showed that the OB context affects the nature of the footprint. Our evidence suggests that KorB is a tetramer. As such, it may be able to bind two sites simultaneously on the same or on different DNA molecules. Using the korABF promoter, which is subject to KorB repression, we demonstrate by footprinting and restriction protection that KorB and RNA polymerase can bind simultaneously. Permanganate footprinting showed that KorB represses this promoter by preventing isomerization of the RNA polymerase-promoter complex from the closed to open form. Images PMID:8464698

  15. Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans.

    PubMed

    Tan, Benedict G; Vijgenboom, Erik; Worrall, Jonathan A R

    2014-01-01

    Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoR(Sl)) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoR(Sl) binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5'-TAC/GTA-3' inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoR(Sl)-DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoR(Sl) tetramers. Finally, differential binding modes may exist in operator sites having more than one 5'-TAC/GTA-3' inverted repeat with implications in vivo for a mechanism of modular control.

  16. The TIE1 transcriptional repressor links TCP transcription factors with TOPLESS/TOPLESS-RELATED corepressors and modulates leaf development in Arabidopsis.

    PubMed

    Tao, Qing; Guo, Dongshu; Wei, Baoye; Zhang, Fan; Pang, Changxu; Jiang, Hao; Zhang, Jinzhe; Wei, Tong; Gu, Hongya; Qu, Li-Jia; Qin, Genji

    2013-02-01

    Leaf size and shape are mainly determined by coordinated cell division and differentiation in lamina. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors are key regulators of leaf development. However, the mechanisms that control TCP activities during leaf development are largely unknown. We identified the TCP Interactor containing EAR motif protein1 (TIE1), a novel transcriptional repressor, as a major modulator of TCP activities during leaf development. Overexpression of TIE1 leads to hyponastic and serrated leaves, whereas disruption of TIE1 causes epinastic leaves. TIE1 is expressed in young leaves and encodes a transcriptional repressor containing a C-terminal EAR motif, which mediates interactions with the TOPLESS (TPL)/TOPLESS-RELATED (TPR) corepressors. In addition, TIE1 physically interacts with CIN-like TCPs. We propose that TIE1 regulates leaf size and morphology by inhibiting the activities of TCPs through recruiting the TPL/TPR corepressors to form a tertiary complex at early stages of leaf development.

  17. bHLH003, bHLH013 and bHLH017 are new targets of JAZ repressors negatively regulating JA responses.

    PubMed

    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

  18. 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

  19. 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.

  20. Crystal structure of omega transcriptional repressor encoded by Streptococcus pyogenes plasmid pSM19035 at 1.5 A resolution.

    PubMed

    Murayama, K; Orth, P; de la Hoz, A B; Alonso, J C; Saenger, W

    2001-12-01

    The 71 amino acid residue omega protein encoded by the Streptococcus pyogenes non-conjugative plasmid pSM19035 is a transcriptional repressor that regulates expression of genes for copy number control and stable maintenance of plasmids. The crystal structure of omega protein has been determined by multiple isomorphous replacement, including anomalous scattering and refined to an R-factor of 21.1 % (R(free)=23.2 %) at 1.5 A resolution. Two monomers related by a non-crystallographic 2-fold axis form a homodimer that occupies the asymmetric unit. Each polypeptide chain is folded into two alpha-helices and one beta-strand forming an antiparallel beta-ribbon in the homodimer. The N-terminal regions (1-23 and 1-22 in subunits I and II, respectively) are not defined in the electron density due to proteolysis of the N-terminal 20 amino acid residues during crystallisation and partial disorder. The omega protein belongs to the structural superfamily of MetJ/Arc repressors featuring a ribbon-helix-helix DNA-binding motif with the beta-ribbon located in and recognizing the major groove of operator DNA; according to a modelled omega protein-DNA complex, residues Arg31 and Arg31' on the beta-ribbon are in positions to interact with a nucleobase, especially guanine. PMID:11733997

  1. Crystal structure of omega transcriptional repressor encoded by Streptococcus pyogenes plasmid pSM19035 at 1.5 A resolution.

    PubMed

    Murayama, K; Orth, P; de la Hoz, A B; Alonso, J C; Saenger, W

    2001-12-01

    The 71 amino acid residue omega protein encoded by the Streptococcus pyogenes non-conjugative plasmid pSM19035 is a transcriptional repressor that regulates expression of genes for copy number control and stable maintenance of plasmids. The crystal structure of omega protein has been determined by multiple isomorphous replacement, including anomalous scattering and refined to an R-factor of 21.1 % (R(free)=23.2 %) at 1.5 A resolution. Two monomers related by a non-crystallographic 2-fold axis form a homodimer that occupies the asymmetric unit. Each polypeptide chain is folded into two alpha-helices and one beta-strand forming an antiparallel beta-ribbon in the homodimer. The N-terminal regions (1-23 and 1-22 in subunits I and II, respectively) are not defined in the electron density due to proteolysis of the N-terminal 20 amino acid residues during crystallisation and partial disorder. The omega protein belongs to the structural superfamily of MetJ/Arc repressors featuring a ribbon-helix-helix DNA-binding motif with the beta-ribbon located in and recognizing the major groove of operator DNA; according to a modelled omega protein-DNA complex, residues Arg31 and Arg31' on the beta-ribbon are in positions to interact with a nucleobase, especially guanine.

  2. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis.

    PubMed

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S; Cuéllar Pérez, Amparo; 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

    2015-08-01

    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

  3. 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

  4. The cox protein of bacteriophage P2 inhibits the formation of the repressor protein and autoregulates the early operon.

    PubMed Central

    Saha, S; Haggård-Ljungquist, E; Nordström, K

    1987-01-01

    The cox gene is the first gene of the early operon of bacteriophage P2. The early promoter Pe and the repressor promoter Pc are located close to each other and in such a way that their transcripts have opposite polarity and show an overlap of about 30 nucleotides. The expression of the early operon and of the C gene was studied in vivo by using fusions to a promoterless cat (chloramphenicol acetyl transferase) gene. The results show that the Cox protein negatively autoregulates the early operon and inhibits the formation of the repressor C. By measuring the efficiency of plating of a series of P2 virulent deletion mutants on bacteria carrying a cloned cox gene, the site of action of the Cox protein was mapped within the Pe-Pc region. The stimulatory effect of the C protein on expression of the Pc promoter was found to be due to inhibition of transcription from Pc; this was demonstrated by mutating Pe which showed that loss of transcription from Pe stimulated transcription from Pc. Hence, this is a case of regulation of gene expression by convergent transcription. By cloning the region C-Pe-Pc-cox such that the cat and kan genes are expressed from Pc and Pe, respectively, it was shown that only one of the resistances (Cm and Km) was expressed. This mimics the choice between lysogeny and lytic growth of the phage. The 'lysogenic' state was very stable whereas the 'lytic' state flipped to the 'lysogenic' at a somewhat higher frequency. The presence of a cloned cox gene drastically stimulated the formation of free phage from a P2-lysogen and dramatically reduced the frequency of lysogenization after P2 infection. We conclude that the pleiotropic effects of the cox (control of excision) gene, namely effects on lysogenization, formation of free phage and site-specific P2 recombination, can be explained by the effect of the Cox protein on the activity of the promoters Pc and Pe. Images Fig. 2. PMID:2826134

  5. 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. PMID:26551668

  6. 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.

  7. The N-terminal domain of the repressor of Staphylococcus aureus phage Φ11 possesses an unusual dimerization ability and DNA binding affinity.

    PubMed

    Biswas, Anindya; Mandal, Sukhendu; Sau, Subrata

    2014-01-01

    Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors. PMID:24747758

  8. The N-Terminal Domain of the Repressor of Staphylococcus aureus Phage Φ11 Possesses an Unusual Dimerization Ability and DNA Binding Affinity

    PubMed Central

    Biswas, Anindya; Mandal, Sukhendu; Sau, Subrata

    2014-01-01

    Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors. PMID:24747758

  9. Genetic Deletion of the Transcriptional Repressor NFIL3 Enhances Axon Growth In Vitro but Not Axonal Repair In Vivo

    PubMed Central

    van der Kallen, Loek R.; Eggers, Ruben; Ehlert, Erich M.; Verhaagen, Joost; Smit, August B.; van Kesteren, Ronald E.

    2015-01-01

    Axonal regeneration after injury requires the coordinated expression of genes in injured neurons. We previously showed that either reducing expression or blocking function of the transcriptional repressor NFIL3 activates transcription of regeneration-associated genes Arg1 and Gap43 and strongly promotes axon outgrowth in vitro. Here we tested whether genetic deletion or dominant-negative inhibition of NFIL3 could promote axon regeneration and functional recovery after peripheral nerve lesion in vivo. Contrary to our expectations, we observed no changes in the expression of regeneration-associated genes and a significant delay in functional recovery following genetic deletion of Nfil3. When NFIL3 function was inhibited specifically in dorsal root ganglia prior to sciatic nerve injury, we observed a decrease in regenerative axon growth into the distal nerve segment rather than an increase. Finally, we show that deletion of Nfil3 changes sciatic nerve lesion-induced expression in dorsal root ganglia of genes that are not typically involved in regeneration, including several olfactory receptors and developmental transcription factors. Together our findings show that removal of NFIL3 in vivo does not recapitulate the regeneration-promoting effects that were previously observed in vitro, indicating that in vivo transcriptional control of regeneration is probably more complex and more robust against perturbation than in vitro data may suggest. PMID:25993115

  10. Regulation of gene expression through a transcriptional repressor that senses acyl-chain length in membrane phospholipids.

    PubMed

    Hofbauer, Harald F; Schopf, Florian H; Schleifer, Hannes; Knittelfelder, Oskar L; Pieber, Bartholomäus; Rechberger, Gerald N; Wolinski, Heimo; Gaspar, Maria L; Kappe, C Oliver; Stadlmann, Johannes; Mechtler, Karl; Zenz, Alexandra; Lohner, Karl; Tehlivets, Oksana; Henry, Susan A; Kohlwein, Sepp D

    2014-06-23

    Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription.

  11. 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.

  12. Genetic Deletion of the Transcriptional Repressor NFIL3 Enhances Axon Growth In Vitro but Not Axonal Repair In Vivo.

    PubMed

    van der Kallen, Loek R; Eggers, Ruben; Ehlert, Erich M; Verhaagen, Joost; Smit, August B; van Kesteren, Ronald E

    2015-01-01

    Axonal regeneration after injury requires the coordinated expression of genes in injured neurons. We previously showed that either reducing expression or blocking function of the transcriptional repressor NFIL3 activates transcription of regeneration-associated genes Arg1 and Gap43 and strongly promotes axon outgrowth in vitro. Here we tested whether genetic deletion or dominant-negative inhibition of NFIL3 could promote axon regeneration and functional recovery after peripheral nerve lesion in vivo. Contrary to our expectations, we observed no changes in the expression of regeneration-associated genes and a significant delay in functional recovery following genetic deletion of Nfil3. When NFIL3 function was inhibited specifically in dorsal root ganglia prior to sciatic nerve injury, we observed a decrease in regenerative axon growth into the distal nerve segment rather than an increase. Finally, we show that deletion of Nfil3 changes sciatic nerve lesion-induced expression in dorsal root ganglia of genes that are not typically involved in regeneration, including several olfactory receptors and developmental transcription factors. Together our findings show that removal of NFIL3 in vivo does not recapitulate the regeneration-promoting effects that were previously observed in vitro, indicating that in vivo transcriptional control of regeneration is probably more complex and more robust against perturbation than in vitro data may suggest.

  13. 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. PMID:27610104

  14. 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

  15. MDM2 Associates with Polycomb Repressor Complex 2 and Enhances Stemness-Promoting Chromatin Modifications Independent of p53.

    PubMed

    Wienken, Magdalena; Dickmanns, Antje; Nemajerova, Alice; Kramer, Daniela; Najafova, Zeynab; Weiss, Miriam; Karpiuk, Oleksandra; Kassem, Moustapha; Zhang, Yanping; Lozano, Guillermina; Johnsen, Steven A; Moll, Ute M; Zhang, Xin; Dobbelstein, Matthias

    2016-01-01

    The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53. PMID:26748827

  16. 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.

  17. Holocarboxylase synthetase acts as a biotin-independent transcriptional repressor interacting with HDAC1, HDAC2 and HDAC7.

    PubMed

    Trujillo-Gonzalez, Isis; Cervantes-Roldan, Rafael; Gonzalez-Noriega, Alfonso; Michalak, Colette; Reyes-Carmona, Sandra; Barrios-Garcia, Tonatiuh; Meneses-Morales, Ivan; Leon-Del-Rio, Alfonso

    2014-03-01

    In human cells, HCS catalyzes the biotinylation of biotin-dependent carboxylases and mediates the transcriptional control of genes involved in biotin metabolism through the activation of a cGMP-dependent signal transduction pathway. HCS also targets to the cell nucleus in association with lamin-B suggesting additional gene regulatory functions. Studies from our laboratory in Drosophila melanogaster showed that nuclear HCS is associated with heterochromatin bands enriched with the transcriptionally repressive mark histone 3 trimethylated at lysine 9. Further, HCS was shown to be recruited to the core promoter of the transcriptionally inactive hsp70 gene suggesting that it may participate in the repression of gene expression, although the mechanism involved remained elusive. In this work, we expressed HCS as a fusion protein with the DNA-binding domain of GAL4 to evaluate its effect on the transcription of a luciferase reporter gene. We show that HCS possesses transcriptional repressor activity in HepG2 cells. The transcriptional function of HCS was shown by in vitro pull down and in vivo co-immunoprecipitation assays to depend on its interaction with the histone deacetylases HDAC1, HDAC2 and HDAC7. We show further that HCS interaction with HDACs and its function in transcriptional repression is not affected by mutations impairing its biotin-ligase activity. We propose that nuclear HCS mediates events of transcriptional repression through a biotin-independent mechanism that involves its interaction with chromatin-modifying protein complexes that include histone deacetylases.

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

    PubMed

    Procházková, Kateřina; Cermáková, Kateřina; Pachl, Petr; Sieglová, Irena; Fábry, Milan; Otwinowski, Zbyszek; Rezáčová, Pavlína

    2012-02-01

    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(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.

  19. A nitrate-inducible GARP family gene encodes an auto-repressible transcriptional repressor in rice.

    PubMed

    Sawaki, Naoya; Tsujimoto, Ryoma; Shigyo, Mikao; Konishi, Mineko; Toki, Seiichi; Fujiwara, Toru; Yanagisawa, Shuichi

    2013-04-01

    Nitrogen is the most important macronutrient in plants and its supply induces responses in gene expression, metabolism and developmental processes. However, the molecular mechanisms underlying the nitrogen responses remain poorly understood. Here we show that the supply of nitrate but not ammonium immediately induces the expression of a transcriptional repressor gene in rice, designated NIGT1 (Nitrate-Inducible, GARP-type Transcriptional Repressor 1). The results of DNA-binding site selection experiments and electrophoretic mobility shift assays indicated that NIGT1 binds to DNA containing either of two consensus sequences, GAATC or GAATATTC. In transient reporter assays, NIGT1 was found to repress transcription from the promoters containing the identified NIGT1-binding sequences in vivo. Furthermore, NIGT1 repressed the activity of its own promoter, suggesting an autorepression mechanism. Consistently, nitrate-induced NIGT1 expression was found to be down-regulated after a transient peak during nitrate treatment, and the nitrate-induced expression of NIGT1 decreased in transgenic rice plants in which this gene was constitutively overexpressed. Furthermore, the chlorophyll content that could be a marker of nitrogen utilization was found to be decreased in NIGT1 overexpressors of rice grown with nitrate medium but not with ammonium medium. Thus, we propose NIGT1 as a nitrate-inducible and autorepressible transcriptional repressor that may play a role in the nitrogen response in rice. Taken together with the fact that the NIGT1-binding sites are conserved in promoter sequences of Arabidopsis NIGT1 homologs, our findings imply the presence of a time-dependent complex system for nitrate-responsive transcriptional regulation that is conserved in both monocots and dicots.

  20. 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.

  1. Mechanism of action of a repressor of dioxin-dependent induction of Cyp1a1 gene transcription.

    PubMed Central

    Watson, A J; Weir-Brown, K I; Bannister, R M; Chu, F F; Reisz-Porszasz, S; Fujii-Kuriyama, Y; Sogawa, K; Hankinson, O

    1992-01-01

    A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions. Images PMID:1314949

  2. 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.

  3. 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

  4. Genomic organization and promoter analysis of a transcriptional repressor gene from Fenneropenaeus chinensis.

    PubMed

    Lai, Xiaofang; Shen, Shanrui; Gao, Huan; Yan, Binlun

    2015-02-01

    In this study, we cloned and sequenced genomic sequences from a Fenneropenaeus chinensis transcriptional repressor gene, FcTR. The FcTR gene is 2,671 bp in length and has four exons and three introns. The 873 bp promoter contains several transcription factor binding sites, including a TATA box and a cyclic AMP-responsive element. Promoter deletion analysis using a luciferase reporter gene identified regulatory elements. Challenge with white spot syndrome virus increased expression from the promoter-deletion constructs. These results suggest that FcTR might play an important role in the shrimp immune response.

  5. Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland.

    PubMed

    Stehle, J H; Foulkes, N S; Molina, C A; Simonneaux, V; Pévet, P; Sassone-Corsi, P

    1993-09-23

    Transcription factor CREM appears to play a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. This axis is modulated by the pineal hormone melatonin, whose production is in turn driven by the endogenous clock. There is striking circadian fluctuation of a novel CREM isoform, ICER, which is expressed at high levels during the night. ICER is generated from an alternative, intronic promoter and functions as a powerful repressor of cyclic AMP-induced transcription. Rhythmic adrenergic signals originated by the clock direct ICER expression by stimulation of the cAMP signal transduction pathway.

  6. The Arabidopsis PR-1 promoter contains multiple integration sites for the coactivator NPR1 and the repressor SNI1.

    PubMed

    Pape, Sebastian; Thurow, Corinna; Gatz, Christiane

    2010-12-01

    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.

  7. 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

  8. 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.

  9. Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor.

    PubMed

    Wiesmeyer, H

    1966-01-01

    Wiesmeyer, Herbert (Vanderbilt University, Nashville, Tenn.). Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor. J. Bacteriol. 91:89-94. 1966.-The concentration of lambda repressor molecules within a lambda lysogenic cell was estimated from the multiplicity of superinfecting homologous phage necessary to permit replication and release of plaque-forming units. A multiplicity of 20 superinfecting phage was found sufficient to permit replication to occur in the normal lambda lysogen. The phage released after lysis of the superinfected lysogen was composed of both prophage and superinfecting phage types. Superinfection of the lysogen at lower multiplicities resulted in the lysis of only a small percentage of infected cells and is thought to represent a possible heterogeneity of repressor concentration in the lysogenic population. Viability of the superinfecting particle was found to be unnecessary for titration of the repressor. The repressor concentration in three lysogens of the nonultraviolet-inducible mutant of lambda, lambda(ind-), was found to be greater than 20 regardless of the host bacterium. However, the number of cells yielding phage after superinfection was found to vary with the particular host. The specificity of the lambda repressor was shown to be limited to homologous phage, as determined following heterologous superinfection experiments with phages T6r, 82c, 434c, 434hy, and 424. In all instances except that of superinfection with phage 434hy, only heterologous phage replication occurred. Superinfection by phage 434hy resulted in the release of both prophage and superinfecting phage types. The latter type represented approximately 80% of the total phage released.

  10. Proton-linked contributions to site-specific interactions of lambda cI repressor and OR.

    PubMed

    Senear, D F; Ackers, G K

    1990-07-17

    The effects of proton activity on the site-specific interactions of cI repressors with operator sites OR were studied by using DNase I footprint titration. Individual-site binding isotherms were obtained for the binding of repressor to each site of wild-type OR and of mutant operators in which binding to some sites is eliminated. The Gibbs energies for binding and for cooperativity (in every operator configuration) were determined at each pH (range 5-8). The proton-linked effects clearly account for a significant fraction of the difference in affinities for the three operator sites. The most dramatic effects on the repressor-operator binding interactions are at acid pH, and therefore do not involve the basic groups in the repressor N-terminal arm known to contact the DNA. Also, the proton-linked effects are different at the three operator sites as indicated by significantly different derivative relationships, partial derivative of ln k versus partial derivative of ln aH = net proton absorption (delta nu bar(H)). These results implicate ionizable repressor groups which may not contact the DNA and conformational differences between the three repressor-operator site complexes as being important components to the mechanism of site specificity. The extensive data base generated by these studies was also used to reevaluate the traditional models used to describe cooperativity in this system. The results confirm the lack of significant cooperative interaction between OR1 and OR3 at all conditions. However, the data for some experimental conditions are clearly inconsistent with the (selection) rule, that cooperative interaction between OR2 and OR3 is eliminated by ligation at OR1.

  11. 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.

  12. Variations in the Fast Folding Rates of the λ-Repressor: A Hybrid Molecular Dynamics Study

    PubMed Central

    Pogorelov, Taras V.; Luthey-Schulten, Zaida

    2004-01-01

    The ability to predict the effects of mutations on protein folding rates and mechanisms would greatly facilitate folding studies. Using a realistic full atom potential coupled with a G\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{{\\bar {o}}}}\\end{equation*}\\end{document}-like potential biased to the native state structure, we have investigated the effects of point mutations on the folding rates of a small single domain protein. The hybrid potential provides a detailed level of description of the folding mechanism that we correlate to features of the folding energy landscapes of fast and slow mutants of an 80-residue-long fragment of the λ-repressor. Our computational reconstruction of the folding events is compared to the recent experimental results of W. Y. Yang and M. Gruebele (see companion article) and T. G. Oas and co-workers on the λ-repressor, and helps to clarify the differences observed in the folding mechanisms of the various mutants. PMID:15240458

  13. Kinetic and thermodynamic studies of purine repressor binding to corepressor and operator DNA.

    PubMed

    Xu, H; Moraitis, M; Reedstrom, R J; Matthews, K S

    1998-04-10

    The kinetic and thermodynamic parameters for purine repressor (PurR)-operator and PurR-guanine binding were determined using fluorescence spectroscopy and nitrocellulose filter binding. Operator binding affinity was increased by the presence of guanine as demonstrated previously (Choi, K. Y., Lu, F., and Zalkin, H. (1994) J. Biol. Chem. 269, 24066-24072; Rolfes, R. J., and Zalkin, H. (1990) J. Bacteriol. 172, 5637-5642), and conversely guanine binding affinity was increased by the presence of operator. Guanine enhanced operator affinity by increasing the association rate constant and decreasing the dissociation rate constant for binding. Operator had minimal effect on the association rate constant for guanine binding; however, this DNA decreased the dissociation rate constant for corepressor by approximately 10-fold. Despite significant sequence and structural similarity between PurR and LacI proteins, PurR binds to its corepressor ligand with a lower association rate constant than LacI binds to its inducer ligand. However, the rate constant for PurR-guanine binding to operator is approximately 3-fold higher than for LacI binding to its cognate operator under the same solution conditions. The distinct metabolic roles of the enzymes under regulation by these two repressor proteins provide a rationale for the observed functional differences.

  14. High-Resolution Specificity from DNA Sequencing Highlights Alternative Modes of Lac Repressor Binding

    PubMed Central

    Zuo, Zheng; Stormo, Gary D.

    2014-01-01

    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. PMID:25209146

  15. 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.

  16. 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

  17. A hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia.

    PubMed

    Ordoñez-Rueda, Diana; Jönsson, Friederike; Mancardi, David A; Zhao, Weidong; Malzac, Aurélie; Liang, Yinming; Bertosio, Elodie; Grenot, Pierre; Blanquet, Véronique; Sabrautzki, Sybille; de Angelis, Martin Hrabě; Méresse, Stéphane; Duprez, Estelle; Bruhns, Pierre; Malissen, Bernard; Malissen, Marie

    2012-09-01

    Using N-ethyl-N-nitrosourea-induced mutagenesis, we established a mouse model with a novel form of neutropenia resulting from a point mutation in the transcriptional repressor Growth Factor Independence 1 (Gfi1). These mice, called Genista, had normal viability and no weight loss, in contrast to mice expressing null alleles of the Gfi1 gene. Furthermore, the Genista mutation had a very limited impact on lymphopoiesis or on T- and B-cell function. Within the bone marrow (BM), the Genista mutation resulted in a slight increase of monopoiesis and in a block of terminal granulopoiesis. This block occurred just after the metamyelocytic stage and resulted in the generation of small numbers of atypical CD11b(+) Ly-6G(int) neutrophils, the nuclear morphology of which resembled that of mature WT neutrophils. Unexpectedly, once released from the BM, these atypical neutrophils contributed to induce mild forms of autoantibody-induced arthritis and of immune complex-mediated lung alveolitis. They additionally failed to provide resistance to acute bacterial infection. Our study demonstrates that a hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia characterized by a split pattern of functional responses, reflecting the distinct thresholds required for eliciting neutrophil-mediated inflammatory and anti-infectious responses.

  18. Transcriptional repressor ZBTB1 promotes chromatin remodeling and translesion DNA synthesis.

    PubMed

    Kim, Hyungjin; Dejsuphong, Donniphat; Adelmant, Guillaume; Ceccaldi, Raphael; Yang, Kailin; Marto, Jarrod A; D'Andrea, Alan D

    2014-04-10

    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 proliferating cell nuclear antigen (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 localizing phospho-KAP-1 to chromatin and enhancing RAD18 accessibility. Collectively, our study implicates a ubiquitin-binding protein in orchestrating chromatin remodeling during DNA repair.

  19. Regulation of Cardiac Gene Expression by KLF15, a Repressor of Myocardin Activity*

    PubMed Central

    Leenders, Joost J.; Wijnen, Wino J.; Hiller, Monika; van der Made, Ingeborg; Lentink, Viola; van Leeuwen, Rick E. W.; Herias, Veronica; Pokharel, Saraswati; Heymans, Stephane; de Windt, Leon J.; Høydal, Morten A.; Pinto, Yigal M.; Creemers, Esther E.

    2010-01-01

    Pathological forms of left ventricular hypertrophy (LVH) often progress to heart failure. Specific transcription factors have been identified that activate the gene program to induce pathological forms of LVH. It is likely that apart from activating transcriptional inducers of LVH, constitutive transcriptional repressors need to be removed during the development of cardiac hypertrophy. Here, we report that the constitutive presence of Krüppel-like factor 15 (KLF15) is lost in pathological hypertrophy and that this loss precedes progression toward heart failure. We show that transforming growth factor-β-mediated activation of p38 MAPK is necessary and sufficient to decrease KLF15 expression. We further show that KLF15 robustly inhibits myocardin, a potent transcriptional activator. Loss of KLF15 during pathological LVH relieves the inhibitory effects on myocardin and stimulates the expression of serum response factor target genes, such as atrial natriuretic factor. This uncovers a novel mechanism where activated p38 MAPK decreases KLF15, an important constitutive transcriptional repressor whose removal seems a vital step to allow the induction of pathological LVH. PMID:20566642

  20. Multiple photomorphogenic repressors work in concert to regulate Arabidopsis seedling development

    PubMed Central

    Dong, Jie; Terzaghi, William; Deng, Xing Wang; Chen, Haodong

    2015-01-01

    Light is both a source of energy and a critically important environmental signal for plant development. Through decades of research, 2 groups of photomorphogenic repressors have been identified. The first group is CONSTITUTIVE PHOTOMORPHOGENIC/DE-ETIOLATED/FUSCA (COP/DET/FUS), which were first identified by genetic screening and then by purification of protein complexes. Another group is the Phytochrome-Interacting Factors (PIFs), which were identified by yeast 2-hybrid screens using phyB as bait. How so many factors work together to repress photomorphogenesis has long been an interesting question. Previously, we demonstrated that CULLIN4 (CUL4) works as a core factor connecting the COP1-SPA complexes, the COP9 signalosome (CSN), and the COP10-DDB1-DET1 (CDD) complex. Recently, we showed that DET1 represses photomorphogenesis through positively regulating the abundance of PIF proteins in the dark. Dr. Huq and his colleagues reported that PIFs may enhance the function of COP1-SPA complexes to promote the degradation of HY5, and thus they synergistically repress photomorphogenesis in the dark. Though much work still needs to be done, these recent breakthroughs shed light on the regulatory relationships among these multiple photomorphogenic repressors. PMID:25853593

  1. 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.

  2. 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.

  3. Antiproliferation activity of a small molecule repressor of liver receptor homolog 1.

    PubMed

    Corzo, Cesar A; Mari, Yelenis; Chang, Mi Ra; Khan, Tanya; Kuruvilla, Dana; Nuhant, Philippe; Kumar, Naresh; West, Graham M; Duckett, Derek R; Roush, William R; Griffin, Patrick R

    2015-02-01

    The orphan nuclear receptor liver receptor homolog 1 (LRH-1; NR5A2) is a potent regulator of cholesterol metabolism and bile acid homeostasis. Recently, LRH-1 has been shown to play an important role in intestinal inflammation and in the progression of estrogen receptor positive and negative breast cancers and pancreatic cancer. Structural studies have revealed that LRH-1 can bind phospholipids and the dietary phospholipid dilauroylphosphatidylcholine activates LRH-1 activity in rodents. Here we characterize the activity of a novel synthetic nonphospholipid small molecule repressor of LRH-1, SR1848 (6-[4-(3-chlorophenyl)piperazin-1-yl]-3-cyclohexyl-1H-pyrimidine-2,4-dione). In cotransfection studies, SR1848 reduced LRH-1-dependent expression of a reporter gene and in cells that endogenously express LRH-1 dose dependently reduced the expression of cyclin-D1 and -E1, resulting in inhibition of cell proliferation. The cellular effects of SR1848 treatment are recapitulated after transfection of cells with small-interfering RNA targeting LRH-1. Immunocytochemistry analysis shows that SR1848 induces rapid translocation of nuclear LRH-1 to the cytoplasm. Combined, these results suggest that SR1848 is a functional repressor of LRH-1 that impacts expression of genes involved in proliferation in LRH-1-expressing cancers. Thus, SR1848 represents a novel chemical scaffold for the development of therapies targeting malignancies driven by LRH-1.

  4. Antiproliferation Activity of a Small Molecule Repressor of Liver Receptor Homolog 1

    PubMed Central

    Corzo, Cesar A.; Mari, Yelenis; Chang, Mi Ra; Khan, Tanya; Kuruvilla, Dana; Nuhant, Philippe; Kumar, Naresh; West, Graham M.; Duckett, Derek R.; Roush, William R.

    2015-01-01

    The orphan nuclear receptor liver receptor homolog 1 (LRH-1; NR5A2) is a potent regulator of cholesterol metabolism and bile acid homeostasis. Recently, LRH-1 has been shown to play an important role in intestinal inflammation and in the progression of estrogen receptor positive and negative breast cancers and pancreatic cancer. Structural studies have revealed that LRH-1 can bind phospholipids and the dietary phospholipid dilauroylphosphatidylcholine activates LRH-1 activity in rodents. Here we characterize the activity of a novel synthetic nonphospholipid small molecule repressor of LRH-1, SR1848 (6-[4-(3-chlorophenyl)piperazin-1-yl]-3-cyclohexyl-1H-pyrimidine-2,4-dione). In cotransfection studies, SR1848 reduced LRH-1-dependent expression of a reporter gene and in cells that endogenously express LRH-1 dose dependently reduced the expression of cyclin-D1 and -E1, resulting in inhibition of cell proliferation. The cellular effects of SR1848 treatment are recapitulated after transfection of cells with small-interfering RNA targeting LRH-1. Immunocytochemistry analysis shows that SR1848 induces rapid translocation of nuclear LRH-1 to the cytoplasm. Combined, these results suggest that SR1848 is a functional repressor of LRH-1 that impacts expression of genes involved in proliferation in LRH-1–expressing cancers. Thus, SR1848 represents a novel chemical scaffold for the development of therapies targeting malignancies driven by LRH-1. PMID:25473120

  5. Ligand efficiency driven design of new inhibitors of Mycobacterium tuberculosis transcriptional repressor EthR using fragment growing, merging, and linking approaches.

    PubMed

    Villemagne, Baptiste; Flipo, Marion; Blondiaux, Nicolas; Crauste, Céline; Malaquin, Sandra; Leroux, Florence; Piveteau, Catherine; Villeret, Vincent; Brodin, Priscille; Villoutreix, Bruno O; Sperandio, Olivier; Soror, Sameh H; Wohlkönig, Alexandre; Wintjens, René; Deprez, Benoit; Baulard, Alain R; Willand, Nicolas

    2014-06-12

    Tuberculosis remains a major cause of mortality and morbidity, killing each year more than one million people. Although the combined use of first line antibiotics (isoniazid, rifampicin, pyrazinamide, and ethambutol) is efficient to treat most patients, the rapid emergence of multidrug resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. Mycobacterial transcriptional repressor EthR is a key player in the control of second-line drugs bioactivation such as ethionamide and has been shown to impair the sensitivity of the human pathogen Mycobacterium tuberculosis to this antibiotic. As a way to identify new potent ligands of this protein, we have developed fragment-based approaches. In the current study, we combined surface plasmon resonance assay, X-ray crystallography, and ligand efficiency driven design for the rapid discovery and optimization of new chemotypes of EthR ligands starting from a fragment. The design, synthesis, and in vitro and ex vivo activities of these compounds will be discussed.

  6. Structure-based functional characterization of repressor of toxin (Rot), a central regulator of staphylococcus aureus virulence

    DOE PAGESBeta

    Killikelly, April; Jakoncic, Jean; Benson, Meredith A.; Ohneck, Elizabeth A.; Sampson, Jared M.; Spurrier, Brett; Torres, Victoer J.; Kong, Xian -Peng

    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

  7. Structure-based functional characterization of repressor of toxin (Rot), a central regulator of Staphylococcus aureus virulence.

    PubMed

    Killikelly, April; Benson, Meredith A; Ohneck, Elizabeth A; Sampson, Jared M; Jakoncic, Jean; Spurrier, Brett; Torres, Victor J; Kong, Xiang-Peng

    2015-01-01

    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 of 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 R(91), at the tip of the wing, plays an important role in DNA binding, likely through interaction with the minor groove. We also found that Y(66), 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. This work provides insight into a precise mechanism by which Rot controls virulence factor regulation in S. aureus.

  8. The CodY pleiotropic repressor controls virulence in gram-positive pathogens.

    PubMed

    Stenz, Ludwig; Francois, Patrice; Whiteson, Katrine; Wolz, Christiane; Linder, Patrick; Schrenzel, Jacques

    2011-07-01

    CodY is involved in the adaptive response to starvation in at least 30 different low G+C gram-positive bacteria. After dimerization and activation by cofactor binding, CodY binds to a consensus palindromic DNA sequence, leading to the repression of approximately 5% of the genome. CodY represses the transcription of target genes when bound to DNA by competition with the RNA polymerase for promoter binding, or by interference with transcriptional elongation as a roadblock. CodY displays enhanced affinity for its DNA target when bound to GTP and/or branched chain amino acids (BCAA). When nutrients become limiting in the postexponential growth phase, a decrease of intracellular levels of GTP and BCAA causes a deactivation of CodY and decreases its affinity for DNA, leading to the induction of its regulon. CodY-regulated genes trigger adaptation of the bacteria to starvation by highly diverse mechanisms, such as secretion of proteases coupled to expression of amino acid transporters, and promotion of survival strategies like sporulation or biofilm formation. Additionally, in pathogenic bacteria, several virulence factors are regulated by CodY. As a function of their access to nutrients, pathogenic gram-positive bacteria express virulence factors in a codY-dependant manner. This is true for the anthrax toxins of Bacillus anthracis and the haemolysins of Staphylococcus aureus. The purpose of this review is to illustrate CodY-regulated mechanisms on virulence in major gram-positive pathogens.

  9. 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. PMID

  10. 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.

  11. A Chlamydia-Specific C-Terminal Region of the Stress Response Regulator HrcA Modulates Its Repressor Activity▿

    PubMed Central

    Chen, Allan L.; Wilson, Adam C.; Tan, Ming

    2011-01-01

    Chlamydial heat shock proteins have important roles in Chlamydia infection and immunopathogenesis. Transcription of chlamydial heat shock genes is controlled by the stress response regulator HrcA, which binds to its cognate operator CIRCE, causing repression by steric hindrance of RNA polymerase. All Chlamydia spp. encode an HrcA protein that is larger than other bacterial orthologs because of an additional, well-conserved C-terminal region. We found that this unique C-terminal tail decreased HrcA binding to CIRCE in vitro as well as HrcA-mediated transcriptional repression in vitro and in vivo. When we isolated HrcA from chlamydiae, we only detected the full-length protein, but we found that endogenous HrcA had a higher binding affinity for CIRCE than recombinant HrcA. To examine this difference further, we tested the effect of the heat shock protein GroEL on the function of HrcA since endogenous chlamydial HrcA has been previously shown to associate with GroEL as a complex. GroEL enhanced the ability of HrcA to bind CIRCE and to repress transcription in vitro, but this stimulatory effect was greater on full-length HrcA than HrcA lacking the C-terminal tail. These findings demonstrate that the novel C-terminal tail of chlamydial HrcA is an inhibitory region and provide evidence that its negative effect on repressor function can be counteracted by GroEL. These results support a model in which GroEL functions as a corepressor that interacts with HrcA to regulate chlamydial heat shock genes. PMID:21965565

  12. 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.

  13. Physical Characterization of the Manganese-sensing Pneumococcal Surface Antigen Repressor (PsaR) from Streptococcus pneumoniae*

    PubMed Central

    Lisher, John P.; Higgins, Khadine A.; Maroney, Michael J.; Giedroc, David P.

    2013-01-01

    Transition metals, including manganese, are required for proper virulence and persistence of many pathogenic bacteria. In Streptococcus pneumoniae (Spn), manganese homeostasis is controlled by a high affinity Mn(II) uptake complex, PsaBCA, and a constitutively expressed efflux transporter, MntE. PsaBCA expression is transcriptionally regulated by the DtxR/MntR family metalloregulatory protein pneumococcal surface antigen repressor (PsaR) in Spn. Here, we present a comprehensive analysis of the metal and DNA-binding properties of PsaR. PsaR is a homodimer in the absence and presence of metals and binds two manganese or zinc per protomer (four per dimer) in two pairs of structurally distinct sites, termed site 1 and site 2. Site 1 is likely filled with Zn(II) in vivo (KZn1≥1013 M−1; KMn1≈108 M−1). The Zn(II)-site 1 complex adopts a pentacoordinate geometry by x-ray absorption spectroscopy containing a single cysteine, and appears analogous to the Cd(II) site observed in S. gordonii ScaR. Site 1 is necessary but not sufficient for full positive allosteric activation of DNA operator binding by metals as measured by ΔGc, the allosteric coupling free energy, since mutants in site 1 show intermediate ΔGc. Site 2 is the primary regulatory site and governs specificity for Mn(II) over Zn(II) in PsaR, with ΔGcZn,Mn>>ΔGcZn,Zn despite the fact that Zn(II) binds site 2 with 40-fold higher affinity relative to Mn(II), i.e., KZn2>KMn2. Mutational studies reveal that Asp7 in site 2 is a critical ligand for Mn(II)-dependent allosteric activation of DNA binding. These findings are discussed in the context of other well-studied DtxR/MntR Mn(II)/Fe(II) metallorepressors. PMID:24067066

  14. The CRE1 carbon catabolite repressor of the fungus Trichoderma reesei: a master regulator of carbon assimilation

    PubMed Central

    2011-01-01

    Background The identification and characterization of the transcriptional regulatory networks governing the physiology and adaptation of microbial cells is a key step in understanding their behaviour. One such wide-domain regulatory circuit, essential to all cells, is carbon catabolite repression (CCR): it allows the cell to prefer some carbon sources, whose assimilation is of high nutritional value, over less profitable ones. In lower multicellular fungi, the C2H2 zinc finger CreA/CRE1 protein has been shown to act as the transcriptional repressor in this process. However, the complete list of its gene targets is not known. Results Here, we deciphered the CRE1 regulatory range in the model cellulose and hemicellulose-degrading fungus Trichoderma reesei (anamorph of Hypocrea jecorina) by profiling transcription in a wild-type and a delta-cre1 mutant strain on glucose at constant growth rates known to repress and de-repress CCR-affected genes. Analysis of genome-wide microarrays reveals 2.8% of transcripts whose expression was regulated in at least one of the four experimental conditions: 47.3% of which were repressed by CRE1, whereas 29.0% were actually induced by CRE1, and 17.2% only affected by the growth rate but CRE1 independent. Among CRE1 repressed transcripts, genes encoding unknown proteins and transport proteins were overrepresented. In addition, we found CRE1-repression of nitrogenous substances uptake, components of chromatin remodeling and the transcriptional mediator complex, as well as developmental processes. Conclusions Our study provides the first global insight into the molecular physiological response of a multicellular fungus to carbon catabolite regulation and identifies several not yet known targets in a growth-controlled environment. PMID:21619626

  15. Identification of Glis1, a novel Gli-related, Kruppel-like zinc finger protein containing transactivation and repressor functions.

    PubMed

    Kim, Yong-Sik; Lewandoski, Mark; Perantoni, Alan O; Kurebayashi, Shogo; Nakanishi, Gen; Jetten, Anton M

    2002-08-23

    In this study, we describe the identification and characterization of a novel Krüppel-like protein named Gli-similar 1 (Glis1). The Glis1 gene encodes an 84.3-kDa proline-rich protein. Its five tandem zinc finger motifs exhibit highest homology with those of members of the Gli and Zic subfamilies of Krüppel-like proteins. Glis1 was mapped to mouse chromosome 4C6. Northern blot analysis showed that expression of the 3.3-kb Glis1 mRNA is most abundant in placenta and adult kidney and expressed at lower levels in testis. Whole mount in situ hybridization on mouse embryos demonstrated that Glis1 is expressed in a temporal and spatial manner during development; expression was most prominent in several defined structures of mesodermal lineage, including craniofacial regions, branchial arches, somites, vibrissal and hair follicles, limb buds, and myotomes. Confocal microscopic analysis showed that Glis1 is localized to the nucleus. The zinc finger region plays an important role in the nuclear localization of Glis1. Electrophoretic mobility shift assays demonstrated that Glis1 is able to bind oligonucleotides containing the Gli-binding site consensus sequence GACCACCCAC. Although monohybrid analysis showed that in several cell types Glis1 was unable to induce transcription of a reporter, deletion mutant analysis revealed the presence of a strong activation function at the carboxyl terminus of Glis1. The activation through this activation function was totally suppressed by a repressor domain at its amino terminus. Constitutively active Ca(2+)-dependent calmodulin kinase IV enhanced Glis1-mediated transcriptional activation about 4-fold and may be mediated by phosphorylation/activation of a co-activator. Our results suggest that Glis1 may play a critical role in the control of gene expression during specific stages of embryonic development.

  16. 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. PMID:26183398

  17. 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.

  18. Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells

    PubMed Central

    Bach, Anne-Sophie; Derocq, Danielle; Laurent-Matha, Valérie; Montcourrier, Philippe; Salwa 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

    2015-01-01

    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. PMID:26183398

  19. 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.

  20. 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

  1. Cooperative non-specific DNA binding by octamerizing lambda cI repressors: a site-specific thermodynamic analysis.

    PubMed

    Pray, T R; Burz, D S; Ackers, G K

    1998-10-01

    Relationships between dimerization and site-specific binding have been characterized previously for wild-type and mutant cI repressors at the right operator (OR) of bacteriophage lambda DNA. However, the roles of higher-order oligomers (tetramers and octamers) that are also formed from these cI molecules have remained elusive. In this study, a clear correlation has been established between repressor oligomerization and non-specific DNA-binding activity. A modification of the quantitative DNase I footprint titration technique has been used to evaluate the degree of saturation of non-specific, OR-flanking lambda DNA by cI repressor oligomers. With the exception of one mutant, only those repressors capable of octamerizing were found to exhibit non-specific DNA-binding activity. The non-specific interaction was accurately modeled using either a one-dimensional, univalent, site-specific Ising lattice approximation, or a more traditional, multivalent lattice approach. It was found that non-specific DNA-binding by repressor oligomers is highly cooperative and energetically independent from site-specific binding at OR. Furthermore, the coupling free energy resolved for non-specific binding was similar to that of site-specific binding for each repressor, suggesting that similar structural elements may mediate the cooperative component of both binding processes. It is proposed that the state of assembly of the repressor molecule modulates its relative affinity for specific and non-specific DNA sequences. These specificities are allosterically regulated by the transmission of assembly-state information from the C-terminal domain, which mediates self-association and cooperativity, to the N-terminal domain, which primarily mediates DNA-binding. While dimers have a high affinity for their cognate sites within OR, tetramers and octamers may preferentially recognize non-specific DNA sequences. The concepts and findings developed in this study may facilitate quantitative

  2. 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.

  3. 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.

  4. 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.

  5. The transcriptional repressor Hes1 attenuates inflammation by regulating transcription elongation.

    PubMed

    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

    2016-08-01

    Most of the known regulatory mechanisms that curb inflammatory gene expression target pre-transcription-initiation steps, and evidence for post-initiation regulation of inflammatory gene expression remains scarce. We found that the transcriptional repressor Hes1 suppressed production of CXCL1, a chemokine that is crucial for recruiting neutrophils. Hes1 negatively regulated neutrophil recruitment in vivo in a manner that was dependent on macrophage-produced CXCL1, and it attenuated the severity of inflammatory arthritis. Mechanistically, inhibition of Cxcl1 expression by Hes1 did not involve modification of transcription initiation. Instead, Hes1 inhibited signal-induced recruitment of the positive transcription-elongation complex P-TEFb and thereby prevented phosphorylation of RNA polymerase II at Ser2 and productive elongation. Thus, our results identify Hes1 as a homeostatic suppressor of inflammatory responses that exerts its suppressive function by regulating transcription elongation.

  6. 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.

  7. Beta-Lactamase Repressor BlaI Modulates Staphylococcus aureus Cathelicidin Antimicrobial Peptide Resistance and Virulence

    PubMed Central

    Pence, Morgan A.; Haste, Nina M.; Meharena, Hiruy S.; Olson, Joshua; Gallo, Richard L.; Nizet, Victor; Kristian, Sascha A.

    2015-01-01

    BlaI is a repressor of BlaZ, the beta-lactamase responsible for penicillin resistance in Staphylococcus aureus. Through screening a transposon library in S. aureus Newman for susceptibility to cathelicidin antimicrobial peptide, we discovered BlaI as a novel cathelicidin resistance factor. Additionally, through integrational mutagenesis in S. aureus Newman and MRSA Sanger 252 strains, we confirmed the role of BlaI in resistance to human and murine cathelidicin and showed that it contributes to virulence in human whole blood and murine infection models. We further demonstrated that BlaI could be a target for innate immune-based antimicrobial therapies; by removing BlaI through subinhibitory concentrations of 6-aminopenicillanic acid, we were able to sensitize S. aureus to LL-37 killing. PMID:26305782

  8. The purine repressor of Bacillus subtilis: a novel combination of domains adapted for transcription regulation.

    PubMed

    Sinha, Sangita C; Krahn, Joseph; Shin, Byung Sik; Tomchick, Diana R; Zalkin, Howard; Smith, Janet L

    2003-07-01

    The purine repressor from Bacillus subtilis, PurR, represses transcription from a number of genes with functions in the synthesis, transport, and metabolism of purines. The 2.2-A crystal structure of PurR reveals a two-domain protein organized as a dimer. The larger C-terminal domain belongs to the PRT structural family, in accord with a sequence motif for binding the inducer phosphoribosylpyrophosphate (PRPP). The PRT domain is fused to a smaller N-terminal domain that belongs to the winged-helix family of DNA binding proteins. A positively charged surface on the winged-helix domain likely binds specific DNA sequences in the recognition site. A second positively charged surface surrounds the PRPP site at the opposite end of the PurR dimer. Conserved amino acids in the sequences of PurR homologs in 21 gram-positive bacteria cluster on the proposed recognition surface of the winged-helix domain and around the PRPP binding site at the opposite end of the molecule, supporting a common function of DNA and PRPP binding for all of the proteins. The structure supports a binding mechanism in which extended regions of DNA interact with extensive protein surface. Unlike most PRT proteins, which are phosphoribosyltransferases (PRTases), PurR lacks catalytic activity. This is explained by a tyrosine side chain that blocks the site for a nucleophile cosubstrate in PRTases. Thus, B. subtilis has adapted an enzyme fold to serve as an effector-binding domain and has used it in a novel combination with the DNA-binding winged-helix domain as a repressor of purine genes.

  9. Oct-1 acts as a transcriptional repressor on the C-reactive protein promoter.

    PubMed

    Voleti, Bhavya; Hammond, David J; Thirumalai, Avinash; Agrawal, Alok

    2012-10-01

    C-reactive protein (CRP), a plasma protein of the innate immune system, is produced by hepatocytes. A critical regulatory region (-42 to -57) on the CRP promoter contains binding site for the IL-6-activated transcription factor C/EBPβ. The IL-1β-activated transcription factor NF-κB binds to a κB site located nearby (-63 to -74). The κB site overlaps an octamer motif (-59 to -66) which is the binding site for the constitutively active transcription factor Oct-1. Oct-1 is known to function both as a transcriptional repressor and as an activator depending upon the promoter context. Also, Oct-1 can regulate gene expression either by binding directly to the promoter or by interacting with other transcription factors bound to the promoter. The aim of this study was to investigate the functions of Oct-1 in regulating CRP expression. In luciferase transactivation assays, overexpressed Oct-1 inhibited (IL-6+IL-1β)-induced CRP expression in Hep3B cells. Deletion of the Oct-1 site from the promoter drastically reduced the cytokine response because the κB site was altered as a consequence of deleting the Oct-1 site. Surprisingly, overexpressed Oct-1 inhibited the residual (IL-6+IL-1β)-induced CRP expression through the promoter lacking the Oct-1 site. Similarly, deletion of the Oct-1 site reduced the induction of CRP expression in response to overexpressed C/EBPβ, and overexpressed Oct-1 inhibited C/EBPβ-induced CRP expression through the promoter lacking the Oct-1 site. We conclude that Oct-1 acts as a transcriptional repressor of CRP expression and it does so by occupying its cognate site on the promoter and also via other transcription factors by an as yet undefined mechanism.

  10. 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

  11. Oligomeric properties and DNA binding specificities of repressor isoforms from the Streptomyces bacteriophage phiC31.

    PubMed

    Wilson, S E; Smith, M C

    1998-05-15

    Three protein isoforms (74, 54 and 42 kDa) are expressed from repressor gene c in the Streptomyces temperate bacteriophage phiC31. Because expression of the two smaller isoforms, 54 and 42 kDa, is sufficient for superinfection immunity, the interaction between these isoforms was studied. The native 42 kDa repressor (Nat42) and an N-terminally 6x histidine-tagged 54 kDa isoform (His54) were shown by co-purification on a Ni-NTA column to interact in Streptomyces lividans . In vitro three repressor preparations, containing Nat42, His54 and the native 54 and 42 kDa isoforms expressed together (Nat54&42), were subjected to chemical crosslinking and gel filtration analysis. Homo- and hetero-tetramers were observed. Previous work showed that the smallest isoform bound to 17 bp operators containing aconservedinvertedrepeat (CIR) and that the CIRs were located at 16 loci throughout the phiC31 genome. One of the CIRs (CIR6) is believed to be critical for regulating the lytic pathway. The DNA binding activities of the three repressor preparations were studied using fragments containing CIRs (CIR3-CIR6) from the essential early region as templates for DNase I footprinting. Whereas Nat42 bound to CIR6, poorly to CIR5 but undetectably to CIR3 or CIR4, the Nat54&42 preparation could bind to all CIRs tested, albeit poorly to CIR3 and CIR4. The His54 isoform bound all CIRs tested. Isoforms expressed from the phiC31 repressor gene, like those which are expressed from many eukaryotic transcription factor genes, apparently have different binding specificities.

  12. Repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K-12: primary structure and identification of the DNA-binding domain.

    PubMed

    Zeng, G; Ye, S; Larson, T J

    1996-12-01

    The nucleotide sequence of the glpEGR operon of Escherichia coli was determined. The translational reading frame at the beginning, middle, and end of each gene was verified. The glpE gene encodes an acidic, cytoplasmic protein of 108 amino acids with a molecular weight of 12,082. The glpG gene encodes a basic, cytoplasmic membrane-associated protein of 276 amino acids with a molecular weight of 31,278. The functions of GlpE and GlpG are unknown. The glpR gene encodes the repressor for the glycerol 3-phosphate regulon, a protein predicted to contain 252 amino acids with a calculated molecular weight of 28,048. The amino acid sequence of the glp repressor was similar to several repressors of carbohydrate catabolic systems, including those of the glucitol (GutR), fucose (FucR), and deoxyribonucleoside (DeoR) systems of E. coli, as well as those of the lactose (LacR) and inositol (IolR) systems of gram-positive bacteria and agrocinopine (AccR) system of Agrobacterium tumefaciens. These repressors constitute a family of related proteins, all of which contain approximately 250 amino acids, possess a helix-turn-helix DNA-binding motif near the amino terminus, and bind a sugar phosphate molecule as the inducing signal. The DNA recognition helix of the glp repressor and the nucleotide sequence of the glp operator were very similar to those of the deo system. The presumptive recognition helix of the glp repressor was changed by site-directed mutagenesis to match that of the deo repressor or, in a separate construct, to abolish DNA binding. Neither altered form of the glp repressor recognized the glp or deo operator, either in vivo or in vitro. However, both altered forms of the glp repressor were negatively dominant to the wild-type glp repressor, indicating that the inability to bind DNA with high affinity was due to alteration of the DNA-binding domain, not to an inability to oligomerize or instability of the altered repressors. For the first time, analysis of repressors

  13. APETALA2 negatively regulates multiple floral organ identity genes in Arabidopsis by recruiting the co-repressor TOPLESS and the histone deacetylase HDA19

    PubMed Central

    Krogan, Naden T.; Hogan, Kendra; Long, Jeff A.

    2012-01-01

    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. PMID:23034631

  14. Repression and activation of transcription by Gal and Lac repressors: involvement of alpha subunit of RNA polymerase.

    PubMed Central

    Choy, H E; Park, S W; Aki, T; Parrack, P; Fujita, N; Ishihama, A; Adhya, S

    1995-01-01

    Gal or Lac repressor binding to an upstream DNA segment, in the absence of DNA looping, represses the P1 promoter located on the same face and activates the P2 promoter situated on the opposite face of the DNA helix in the gal operon. Both inhibition and stimulation of transcription requires the physical presence of the C-terminal domain of the alpha subunit of RNA polymerase although the latter is not required for transcription itself. We propose that Gal and Lac repressors inhibit or stimulate transcription initiation by disabling or stimulating RNA polymerase activity at a post-binding step by directly or indirectly altering the C-terminal alpha domain to an unfavorable state at P1 or a more favorable state at P2, respectively. Images PMID:7556095

  15. 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°.

  16. Rett syndrome mutations abolish the interaction of MeCP2 with the NCoR/SMRT co-repressor.

    PubMed

    Lyst, Matthew J; Ekiert, Robert; Ebert, Daniel H; Merusi, Cara; Nowak, Jakub; Selfridge, Jim; Guy, Jacky; Kastan, Nathaniel R; Robinson, Nathaniel D; de Lima Alves, Flavia; Rappsilber, Juri; Greenberg, Michael E; Bird, Adrian

    2013-07-01

    Rett syndrome (RTT) is a severe neurological disorder that is caused by mutations in the MECP2 gene. Many missense mutations causing RTT are clustered in the DNA-binding domain of MeCP2, suggesting that association with chromatin is critical for its function. We identified a second mutational cluster in a previously uncharacterized region of MeCP2. We found that RTT mutations in this region abolished the interaction between MeCP2 and the NCoR/SMRT co-repressor complexes. Mice bearing a common missense RTT mutation in this domain exhibited severe RTT-like phenotypes. Our data are compatible with the hypothesis that brain dysfunction in RTT is caused by a loss of the MeCP2 'bridge' between the NCoR/SMRT co-repressors and chromatin.

  17. TG-interacting factor 1 acts as a transcriptional repressor of sterol O-acyltransferase 2[S

    PubMed Central

    Pramfalk, Camilla; Melhuish, Tiffany A.; Wotton, David; Jiang, Zhao-Yan; Eriksson, Mats; Parini, Paolo

    2014-01-01

    Acat2 [gene name: sterol O-acyltransferase 2 (SOAT2)] esterifies cholesterol in enterocytes and hepatocytes. This study aims to identify repressor elements in the human SOAT2 promoter and evaluate their in vivo relevance. We identified TG-interacting factor 1 (Tgif1) to function as an important repressor of SOAT2. Tgif1 could also block the induction of the SOAT2 promoter activity by hepatocyte nuclear factor 1α and 4α. Women have ∼30% higher hepatic TGIF1 mRNA compared with men. Depletion of Tgif1 in mice increased the hepatic Soat2 expression and resulted in higher hepatic lipid accumulation and plasma cholesterol levels. Tgif1 is a new player in human cholesterol metabolism. PMID:24478032

  18. Comparison of operator-specific and nonspecific DNA binding of the lambda cI repressor: [KCl] and pH effects.

    PubMed

    Senear, D F; Batey, R

    1991-07-01

    The effects of proton and KCl activity on the nonspecific lambda cI repressor-DNA interactions and on the site-specific repressor-O(R) interactions were compared, in order to assess their roles in site specificity. The repressor-O(R) interactions were studied by using DNase I footprint titration. The Gibbs free energy changes for binding and for cooperativity were determined between 25 and 300 mM KCl, from individual-site isotherms for the binding of repressor to O(R) and to reduced-valency mutants. The proton-linked effects on repressor-O(R) interactions have been published [Senear, D. F., & Ackers, G. K. (1990) Biochemistry 29, 6568-6577; Senear, D. F., & Bolen, D. W. (1991) Methods Enzymol. (in press)]. Nonspecific binding was studied by using a nitrocellulose filter binding assay, which proved advantageous in this case, due to the relatively weak nonspecific binding, and precipitation of repressor-DNA complexes. Filter binding provided measurements at low binding density where precipitation did not occur. The data provide estimates of the Gibbs free energy changes for nonspecific, intrinsic binding, but not for cooperativity. The KCl concentration dependencies of the intrinsic binding constants indicate that ion release plays similar roles in distinguishing between the operators and in discriminating operator from nonoperator DNA. Binding to DNA is accompanied by net proton absorption. Near neutral pH, proton linkages to operator and nonoperator binding are the same. Differences at acid and at basic pH implicate the same ionizable repressor groups in distinguishing between the operators and in discriminating operator from nonoperator DNA. The results indicate similar overall modes of operator and nonoperator binding of repressor, but implicate indirect effects of DNA sequence as important contributors to sequence recognition.

  19. Expression and functions of the repressor element 1 (RE-1)-silencing transcription factor (REST) in breast cancer.

    PubMed

    Lv, Hui; Pan, Guoqing; Zheng, Guopei; Wu, Xiaoying; Ren, Hongzheng; Liu, Ying; Wen, Jifang

    2010-07-01

    The repressor element 1 (RE-1)-silencing transcription factor (REST), also known as the neuron-restrictive silencer factor (NRSF) or repressor binding to the X2 box (XBR), REST/NRSF/XBR, is originally discovered as a transcriptional repressor of a large number of primarily terminal neuronal differentiation genes in non-neuronal cells and neural stem cells (NSCs). Recently, the tumor-suppressor function of REST is finally proved. However, the expression profile and function of REST in breast cancer are not very clear. In this study, the expression of REST was detected in breast cancer tissue by immunohistochemistry. The results showed that REST expression was significantly lower in breast cancer samples compared to normal and benign breast samples (P < 0.05). Furthermore, the shRNA approach was used to investigate the function of REST in human breast cancer cells. Knocking down REST expression by shRNA in the human breast cancer MCF-7 cells resulted in an increase in cell proliferation, suppression in apoptosis, and reduced sensitivity to anticancer drug with a concurrent significantly up-regulated expression of Bcl-2. These data implied a significant role of REST in breast cancer. The reduced expression of REST might contribute to the breast cancer pathogenesis. PMID:20564196

  20. 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.

  1. 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

  2. Cyclic AMP-dependent constitutive expression of gal operon: use of repressor titration to isolate operator mutations.

    PubMed Central

    Irani, M; Orosz, L; Busby, S; Taniguchi, T; Adhya, S

    1983-01-01

    When the gal operator region is present in a multicopy plasmid it binds to all ("titrates") the gal repressor and "induces" the chromosomal gal operon. To make operator mutations (Oa) with reduced affinity toward the repressor, plasmid DNA was irradiated with UV light and mutant derivatives were isolated that were unable to release the chromosomal gal genes from repression. Then with such an Oa plasmid operator revertants were isolated that had reacquired the ability to release repression. Both sets of mutations have been localized by DNA sequence analysis. When the Oa mutations were transferred from the plasmid to the chromosome by recombination these mutant operators were found to make gal expression constitutive (independent of repressor) but still dependent on cAMP, whereas the previously reported gal operator mutants (Oc) are constitutive both in the presence and in the absence of cAMP. The titration method of isolating mutants enables the isolation of strains with operator mutations that also affect normal promoter activity, and it provides an easy way to isolate revertants of operator mutations. Images PMID:6308647

  3. Regulation of RNA splicing by the methylation-dependent transcriptional repressor methyl-CpG binding protein 2

    PubMed Central

    Young, Juan I.; Hong, Eugene P.; Castle, John C.; Crespo-Barreto, Juan; Bowman, Aaron B.; Rose, Matthew F.; Kang, Dongcheul; Richman, Ron; Johnson, Jason M.; Berget, Susan; Zoghbi, Huda Y.

    2005-01-01

    Rett syndrome (RTT) is a postnatal neurodevelopmental disorder characterized by the loss of acquired motor and language skills, autistic features, and unusual stereotyped movements. RTT is caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). Mutations in MECP2 cause a variety of neurodevelopmental disorders including X-linked mental retardation, psychiatric disorders, and some cases of autism. Although MeCP2 was identified as a methylation-dependent transcriptional repressor, transcriptional profiling of RNAs from mice lacking MeCP2 did not reveal significant gene expression changes, suggesting that MeCP2 does not simply function as a global repressor. Changes in expression of a few genes have been observed, but these alterations do not explain the full spectrum of Rett-like phenotypes, raising the possibility that additional MeCP2 functions play a role in pathogenesis. In this study, we show that MeCP2 interacts with the RNA-binding protein Y box-binding protein 1 and regulates splicing of reporter minigenes. Importantly, we found aberrant alternative splicing patterns in a mouse model of RTT. Thus, we uncovered a previously uncharacterized function of MeCP2 that involves regulation of splicing, in addition to its role as a transcriptional repressor. PMID:16251272

  4. 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

  5. Repressor and activator protein accelerates hepatic ischemia reperfusion injury by promoting neutrophil inflammatory response

    PubMed Central

    Li, Chang Xian; Lo, Chung Mau; Lian, Qizhou; Ng, Kevin Tak-Pan; Liu, Xiao Bing; Ma, Yuen Yuen; Qi, Xiang; Yeung, Oscar Wai Ho; Tergaonkar, Vinay; Yang, Xin Xiang; Liu, Hui; Liu, Jiang; Shao, Yan; Man, Kwan

    2016-01-01

    Repressor and activator protein (Rap1) directly regulates nuclear factor-κB (NF-κB) dependent signaling, which contributes to hepatic IRI. We here intended to investigate the effect of Rap1 in hepatic ischemia reperfusion injury (IRI) and to explore the underlying mechanisms. The association of Rap1 expression with hepatic inflammatory response were investigated in both human and rat liver transplantation. The effect of Rap1 in hepatic IRI was studied in Rap1 knockout mice IRI model in vivo and primary cells in vitro. Our results showed that over expression of Rap1 was associated with severe liver graft inflammatory response, especially in living donor liver transplantation. The results were also validated in rat liver transplantation model. In mice hepatic IRI model, the knockout of Rap1 reduced hepatic damage and hepatic inflammatory response. In primary cells, the knockout of Rap1 suppressed neutrophils migration activity and adhesion in response to liver sinusoidal endothelial cells through down-regulating neutrophils F-Actin expression and CXCL2/CXCR2 pathway. In addition, the knockout of Rap1 also decreased production of pro-inflammatory cytokines/chemokines in primary neutrophils and neutrophils-induced hepatocyte damage. In conclusion, Rap1 may induce hepatic IRI through promoting neutrophils inflammatory response. Rap1 may be the potential therapeutic target of attenuating hepatic IRI. PMID:27050284

  6. Cytotype regulation of P transposable elements in Drosophila melanogaster: repressor polypeptides or piRNAs?

    PubMed

    Jensen, Philip A; Stuart, Jeremy R; Goodpaster, Michael P; Goodman, Joseph W; Simmons, Michael J

    2008-08-01

    The telomeric P elements TP5 and TP6 are associated with the P cytotype, a maternally inherited condition that represses P-element-induced hybrid dysgenesis in the Drosophila germ line. To see if cytotype repression by TP5 and TP6 might be mediated by the polypeptides they could encode, hobo transgenes carrying these elements were tested for expression of mRNA in the female germ line and for repression of hybrid dysgenesis. The TP5 and TP6 transgenes expressed more germ-line mRNA than the native telomeric P elements, but they were decidedly inferior to the native elements in their ability to repress hybrid dysgenesis. These paradoxical results are inconsistent with the repressor polypeptide model of cytotype. An alternative model based on the destruction of P transposase mRNA by Piwi-interacting (pi) RNAs was supported by finding reduced P mRNA levels in flies that carried the native telomeric P elements, which are inserted in a known major piRNA locus. PMID:18579507

  7. 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

  8. 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

  9. Morpholino antisense oligonucleotides targeting intronic repressor Element1 improve phenotype in SMA mouse models

    PubMed Central

    Osman, Erkan Y.; Miller, Madeline R.; Robbins, Kate L.; Lombardi, Abby M.; Atkinson, Arleigh K.; Brehm, Amanda J.; Lorson, Christian L.

    2014-01-01

    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 (E1MO-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, E1MO-ASO treatment in an intermediate SMA mouse (SMNRT 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. PMID:24781211

  10. The transcriptional repressor EUO regulates both subsets of Chlamydia late genes.

    PubMed

    Rosario, Christopher J; Hanson, Brett R; Tan, Ming

    2014-11-01

    The pathogenic bacterium Chlamydia replicates in a eukaryotic host cell via a developmental cycle marked by temporal waves of gene expression. We have previously shown that late genes transcribed by the major chlamydial RNA polymerase, σ(66) RNA polymerase, are regulated by a transcriptional repressor EUO. We now report that EUO also represses promoters for a second subset of late genes that are transcribed by an alternative polymerase called σ(28) RNA polymerase. EUO bound in the vicinity of six σ(28) -dependent promoters and inhibited transcription of each promoter. We used a mutational analysis to demonstrate that the EUO binding site functions as an operator that is necessary and sufficient for EUO-mediated repression of σ(28) -dependent transcription. We also verified specific binding of EUO to σ(66) -dependent and σ(28) -dependent promoters with a DNA immunoprecipitation assay. These findings support a model in which EUO represses expression of both σ(66) -dependent and σ(28) -dependent late genes. We thus propose that EUO is the master regulator of late gene expression in the chlamydial developmental cycle.

  11. Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking.

    PubMed

    Futran, Alan S; Kyin, Saw; Shvartsman, Stanislav Y; Link, A James

    2015-07-14

    Extracellular signal-regulated kinase (ERK) coordinates cellular responses to a range of stimuli by phosphorylating its numerous substrates. One of these substrates, Capicua (Cic), is a transcriptional repressor that was first identified in Drosophila and has been implicated in a number of human diseases. Here we use a chemical biology approach to map the binding interface of ERK and Cic. The noncanonical amino acid p-azidophenylalanine (AzF) was introduced into the ERK-binding region of Drosophila Cic, and photocrosslinking and tandem mass spectrometry were used to pinpoint its binding site on ERK. We also identified the ERK-binding region of human Cic and showed that it binds to the same site on ERK despite lacking conservation with the Drosophila Cic binding region. Finally, we mapped the amino acids involved in human Cic binding to ERK using AzF-labeled ERK. These results reveal the molecular details of the ERK-Cic interaction and demonstrate that the photocrosslinking approach is complementary to existing methods for mapping kinase-substrate binding interfaces.

  12. The Transcriptional Repressor ZNF503/Zeppo2 Promotes Mammary Epithelial Cell Proliferation and Enhances Cell Invasion.

    PubMed

    Shahi, Payam; Slorach, Euan M; Wang, Chih-Yang; Chou, Jonathan; Lu, Angela; Ruderisch, Aline; Werb, Zena

    2015-02-01

    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.

  13. NLRP7, Involved in Hydatidiform Molar Pregnancy (HYDM1), Interacts with the Transcriptional Repressor ZBTB16.

    PubMed

    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.

  14. ZBTB20 is a sequence-specific transcriptional repressor of alpha-fetoprotein gene.

    PubMed

    Zhang, Hai; Cao, Dongmei; Zhou, Luting; Zhang, Ye; Guo, Xiaoqin; Li, Hui; Chen, Yuxia; Spear, Brett T; Wu, Jia-Wei; Xie, Zhifang; Zhang, Weiping J

    2015-07-15

    Alpha-fetoprotein (AFP) represents a classical model system to study developmental gene regulation in mammalian cells. We previously reported that liver ZBTB20 is developmentally regulated and plays a central role in AFP postnatal repression. Here we show that ZBTB20 is a sequence-specific transcriptional repressor of AFP. By ELISA-based DNA-protein binding assay and conventional gel shift assay, we successfully identified a ZBTB20-binding site at -104/-86 of mouse AFP gene, flanked by two HNF1 sites and two C/EBP sites in the proximal promoter. Importantly, mutation of the core sequence in this site fully abolished its binding to ZBTB20 in vitro, as well as the repression of AFP promoter activity by ZBTB20. The unique ZBTB20 site was highly conserved in rat and human AFP genes, but absent in albumin genes. These help to explain the autonomous regulation of albumin and AFP genes in the liver after birth. Furthermore, we demonstrated that transcriptional repression of AFP gene by ZBTB20 was liver-specific. ZBTB20 was dispensable for AFP silencing in other tissues outside liver. Our data define a cognate ZBTB20 site in AFP promoter which mediates the postnatal repression of AFP gene in the liver.

  15. The transcriptional repressor BLIMP1 curbs host defenses by suppressing expression of the chemokine CCL8.

    PubMed

    Severa, Martina; Islam, Sabina A; Waggoner, Stephen N; Jiang, Zhaozhao; Kim, Nancy D; Ryan, Glennice; Kurt-Jones, Evelyn; Charo, Israel; Caffrey, Daniel R; Boyartchuk, Victor L; Luster, Andrew D; Fitzgerald, Katherine A

    2014-03-01

    The transcriptional repressor B lymphocyte-induced maturation protein 1 (BLIMP1) is a master regulator of B and T cell differentiation. To examine the role of BLIMP1 in innate immunity, we used a conditional knockout (CKO) of Blimp1 in myeloid cells and found that Blimp1 CKO mice were protected from lethal infection induced by Listeria monocytogenes. Transcriptome analysis of Blimp1 CKO macrophages identified the murine chemokine (C-C motif) ligand 8, CCL8, as a direct target of Blimp1-mediated transcriptional repression in these cells. BLIMP1-deficient macrophages expressed elevated levels of Ccl8, and consequently Blimp1 CKO mice had higher levels of circulating CCL8, resulting in increased neutrophils in the peripheral blood, promoting a more aggressive antibacterial response. Mice lacking the Ccl8 gene were more susceptible to L. monocytogenes infection than were wild-type mice. Although CCL8 failed to recruit neutrophils directly, it was chemotactic for γ/δ T cells, and CCL8-responsive γ/δ T cells were enriched for IL-17F. Finally, CCL8-mediated enhanced clearance of L. monocytogenes was dependent on γ/δ T cells. Collectively, these data reveal an important role for BLIMP1 in modulating host defenses by suppressing expression of the chemokine CCL8.

  16. The Mycobacterium tuberculosis transcriptional repressor EthR is negatively regulated by Serine/Threonine phosphorylation.

    PubMed

    Leiba, Jade; Carrère-Kremer, Séverine; Blondiaux, Nicolas; Dimala, Martin Moune; Wohlkönig, Alexandre; Baulard, Alain; Kremer, Laurent; Molle, Virginie

    2014-04-18

    Recent efforts have underlined the role of Serine/Threonine Protein Kinases (STPKs) in growth, pathogenesis and cell wall metabolism in mycobacteria. Herein, we demonstrated that the Mycobacterium tuberculosis EthR, a transcriptional repressor that regulates the activation process of the antitubercular drug ethionamide (ETH) is a specific substrate of the mycobacterial kinase PknF. ETH is a prodrug that must undergo bioactivation by the monooxygenease EthA to exert its antimycobacterial activity and previous studies reported that EthR represses transcription of ethA by binding to the ethA-ethR intergenic region. Mass spectrometry analyses and site-directed mutagenesis identified a set of four phosphoacceptors, namely Thr2, Thr3, Ser4 and Ser7. This was further supported by the complete loss of PknF-dependent phosphorylation of a phosphoablative EthR mutant protein. Importantly, a phosphomimetic version of EthR, in which all phosphosites were replaced by Asp residues, exhibited markedly decreased DNA-binding activity compared with the wild-type protein. Together, these findings are the first demonstration of EthR phosphorylation and indicate that phosphorylation negatively affects its DNA-binding activity, which may impact ETH resistance levels in M. tb.

  17. Transcriptional Repressor DAXX Promotes Prostate Cancer Tumorigenicity via Suppression of Autophagy.

    PubMed

    Puto, Lorena A; Brognard, John; Hunter, Tony

    2015-06-19

    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 Oncomine(TM) 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.

  18. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements.

    PubMed

    Thakore, Pratiksha I; D'Ippolito, Anthony M; Song, Lingyun; Safi, Alexias; Shivakumar, Nishkala K; Kabadi, Ami M; Reddy, Timothy E; Crawford, Gregory E; Gersbach, Charles A

    2015-12-01

    Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.

  19. Tumor repressor protein 53 and steroid hormones provide a new paradigm for ovarian cancer metastases.

    PubMed

    Mullany, Lisa K; Liu, Zhilin; Wong, Kwong-Kwok; Deneke, Victoria; Ren, Yi Athena; Herron, Alan; Richards, JoAnne S

    2014-01-01

    The functional status of the tumor repressor protein (TP53 or TRP53) is a defining feature of ovarian cancer. Mutant or null alleles of TP53 are expressed in greater than 90% of all high-grade serous adenocarcinomas. Wild-type TP53 is elevated in low-grade serous adenocarcinomas in women and in our Pten;Kras;Amhr2-Cre mutant mouse model. Disruption of the Trp53 gene in this mouse model did not lead to high-grade ovarian cancer but did increase expression of estrogen receptor α (ESR1) and markedly enhanced the responsiveness of these cells to estrogen. Specifically, when Trp53-positive and Trp53 null mutant mice were treated with estradiol or vehicle, only the Trp53 null and Esr1-positive tumors respond vigorously to estradiol in vivo and exhibit features characteristic of high-grade type ovarian cancer: invasive growth into the ovarian stroma, rampant metastases to the peritoneal cavity, and nuclear atypia. Estrogen promoted and progesterone suppressed the growth of Trp53 null ovarian tumors and tumor cells injected ip, sc, or when grown in matrigel. Exposure of the Trp53 depleted cells to estrogen also has a profound impact on the tumor microenvironment and immune-related events. These results led to the new paradigm that TRP53 status is related to the susceptibility of transformed ovarian surface epithelial cells to estradiol-induced metastases and nuclear atypia via increased levels of estradiol receptor α.

  20. A testis cytoplasmic RNA-binding protein that has the properties of a translational repressor.

    PubMed Central

    Lee, K; Fajardo, M A; Braun, R E

    1996-01-01

    Translation of the mouse protamine 1 (Prm-1) mRNA is repressed for several days during male germ cell differentiation. With the hope of cloning genes that regulate the translational repression of Prm-1, we screened male germ cell cDNA expression libraries with the 3' untranslated region of the Prm-1 RNA. From this screen we obtained two independent clones that encode Prbp, a Prm-1 RNA-binding protein. Prbp contains two copies of a double-stranded-RNA-binding domain. In vitro, the protein binds to a portion of the Prm-1 3' untranslated region previously shown to be sufficient for translational repression in transgenic mice, as well as to poly(I). poly(C). Prbp protein is present in multiple forms in cytoplasmic extracts prepared from wild-type mouse testes and is absent from testes of germ cell-deficient mouse mutants, suggesting that Prbp is restricted to the germ cells of the testis. Immunocytochemical localization confirmed that Prbp is present in the cytoplasmic compartment of late-stage meiotic cells and haploid round spermatids. Recombinant Prbp protein inhibits the translation of multiple mRNAs in a wheat germ lysate, suggesting that Prbp acts to repress translation in round spermatids. While this protein lacks complete specificity for Prm-1-containing RNAs in vitro, the properties of Prbp are consistent with it acting as a general repressor of translation. PMID:8649414

  1. 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.

  2. Loss of the RNA polymerase III repressor MAF1 confers obesity resistance

    PubMed Central

    Bonhoure, Nicolas; Byrnes, Ashlee; Moir, Robyn D.; Hodroj, Wassim; Preitner, Frédéric; Praz, Viviane; Marcelin, Genevieve; Chua, Streamson C.; Martinez-Lopez, Nuria; Singh, Rajat; Moullan, Norman; Auwerx, Johan; Willemin, Gilles; Shah, Hardik; Hartil, Kirsten; Vaitheesvaran, Bhavapriya; Kurland, Irwin

    2015-01-01

    MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1−/− mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1−/− mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD+, and is associated with obesity resistance. Consistent with this, NAD+ levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences. PMID:25934505

  3. Differential signal transduction via TrmB, a sugar sensing transcriptional repressor of Pyrococcus furiosus.

    PubMed

    Lee, Sung-Jae; Surma, Melanie; Seitz, Sabine; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2007-06-01

    TrmB is a transcriptional repressor of the hyperthermophilic archaeon Pyrococcus furiosus serving at least two operons. TrmB represses genes encoding an ABC transporter for trehalose and maltose (the TM system) with trehalose and maltose as inducers. TrmB also represses genes encoding another ABC transporter for maltodextrins (the MD system) with maltotriose and sucrose as inducers. Here we report that glucose which was also bound by TrmB acted as a corepressor (causing stronger repression) for both the TM and the MD system. Binding of glucose by TrmB was increased in the presence of TM promoter DNA. Maltose which acted as inducer for the TM system acted as a corepressor for the MD system intensifying repression. We propose that the differential conformational changes of TrmB in response to binding the different sugars governs the ability of TrmB to interact with the promoter region and represents a simple mechanism for selecting the usage of one carbon source over the other, reminiscent of catabolite repression in bacteria.

  4. SPBP Is a Phosphoserine-Specific Repressor of Estrogen Receptor α

    PubMed Central

    Gburcik, Valentina; Bot, Nathalie; Maggiolini, Marcello; Picard, Didier

    2005-01-01

    Multiple signaling pathways stimulate the activity of estrogen receptor α (ERα) by direct phosphorylation within its N-terminal activation function 1 (AF1). How phosphorylation affects AF1 activity remains poorly understood. We performed a phage display screen for human proteins that are exclusively recruited to the phosphorylated form of AF1 and found the stromelysin-1 platelet-derived growth factor-responsive element-binding protein (SPBP). In a purified system, SPBP bound only the in vitro-phosphorylated form of the ERα AF1 or the phosphoserine mimic S118E, and the interaction domain could be mapped to a 42-amino-acid fragment of SPBP. In cells, SPBP preferentially interacted with liganded and phosphorylated ERα. Functionally, SPBP behaved as a repressor of activated ERα, which extends its previously demonstrated roles as a DNA binding transactivation factor and coactivator of other transcription factors. By targeting the phosphorylated form of AF1, SPBP may contribute to attenuating and fine-tuning ERα activity. A functional consequence is that SPBP inhibits the proliferation of ERα-dependent but not ERα-independent breast cancer cell lines, mirroring a reported negative correlation with the ERα status of breast tumors. PMID:15831449

  5. SPBP is a phosphoserine-specific repressor of estrogen receptor alpha.

    PubMed

    Gburcik, Valentina; Bot, Nathalie; Maggiolini, Marcello; Picard, Didier

    2005-05-01

    Multiple signaling pathways stimulate the activity of estrogen receptor alpha (ERalpha) by direct phosphorylation within its N-terminal activation function 1 (AF1). How phosphorylation affects AF1 activity remains poorly understood. We performed a phage display screen for human proteins that are exclusively recruited to the phosphorylated form of AF1 and found the stromelysin-1 platelet-derived growth factor-responsive element-binding protein (SPBP). In a purified system, SPBP bound only the in vitro-phosphorylated form of the ERalpha AF1 or the phosphoserine mimic S118E, and the interaction domain could be mapped to a 42-amino-acid fragment of SPBP. In cells, SPBP preferentially interacted with liganded and phosphorylated ERalpha. Functionally, SPBP behaved as a repressor of activated ERalpha, which extends its previously demonstrated roles as a DNA binding transactivation factor and coactivator of other transcription factors. By targeting the phosphorylated form of AF1, SPBP may contribute to attenuating and fine-tuning ERalpha activity. A functional consequence is that SPBP inhibits the proliferation of ERalpha-dependent but not ERalpha-independent breast cancer cell lines, mirroring a reported negative correlation with the ERalpha status of breast tumors. PMID:15831449

  6. A knockdown with smoke model reveals FHIT as a repressor of Heme oxygenase 1

    PubMed Central

    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. PMID:25486479

  7. Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements

    PubMed Central

    Thakore, Pratiksha I.; D’Ippolito, Anthony M; Song, Lingyun; Safi, Alexias; Shivakumar, Nishkala K.; Kabadi, Ami M.; Reddy, Timothy E.; Crawford, Gregory E.; Gersbach, Charles A.

    2015-01-01

    Epigenome editing with the CRISPR/Cas9 platform is a promising technology to modulate gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of the nuclease-inactive dCas9 to the KRAB repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB is not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates expression of multiple globin genes. Genome-wide analyses demonstrated that localization of dCas9-KRAB to HS2 specifically induced H3K9 tri-methylation (H3K9me3) at the enhancer and reduced the chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome. PMID:26501517

  8. KHDC1A, a Novel Translational Repressor, Induces Endoplasmic Reticulum-Dependent Apoptosis

    PubMed Central

    Cai, Congli; Liu, Jing; Wang, Chao

    2012-01-01

    RNA binding proteins are characterized as a new family of apoptosis inducers; however, the mechanism by which they induce apoptosis is poorly understood. KHDC1 family members were recently identified as K-homology (KH)-domain containing RNA binding proteins that are unique to eutherian mammals and highly expressed in oocytes. In this study, we report that the expression of KHDC1A induces caspase-3 dependent apoptosis and inhibits mRNA translation, and the translational repression is independent of apoptosis. We demonstrate that both the N-terminus and C-terminus of KHDC1A are required for its pro-apoptotic and translational repression activities. Furthermore, in the C-terminus of KHDC1A, a putative trans-membrane motif (TMM) is critical for these activities. In addition, the ectopically expressed KHDC1A is localized to the endoplasmic reticulum (ER) and changes the morphology of the ER. The inhibition of ER-specific caspase-12 successfully rescues KHDC1A-induced apoptosis, but not Fas-induced apoptosis. Taken together, we conclude that KHDC1A functions as a global translational repressor and induces apoptosis through an ER-dependent signaling pathway. PMID:22731819

  9. Acidic pH promotes oligomerization and membrane insertion of the BclXL apoptotic repressor.

    PubMed

    Bhat, Vikas; Kurouski, Dmitry; Olenick, Max B; McDonald, Caleb B; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Lednev, Igor K; Farooq, Amjad

    2012-12-01

    Solution pH is believed to serve as an intricate regulatory switch in the induction of apoptosis central to embryonic development and cellular homeostasis. Herein, using an array of biophysical techniques, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megadalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues. PMID:22960132

  10. 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.

  11. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2

    PubMed Central

    Heyne, Kristina; Förster, Juliane; Schüle, Roland; Roemer, Klaus

    2014-01-01

    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. PMID:24413661

  12. Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System.

    PubMed

    Mettrick, Karla A; Lawrence, Nikki; Mason, Claire; Weaver, Georgia M; Corocher, Tayla-Ann; Grainge, Ian

    2016-01-01

    Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell's replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system. PMID:27583408

  13. Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

    PubMed

    Bonhoure, Nicolas; Byrnes, Ashlee; Moir, Robyn D; Hodroj, Wassim; Preitner, Frédéric; Praz, Viviane; Marcelin, Genevieve; Chua, Streamson C; Martinez-Lopez, Nuria; Singh, Rajat; Moullan, Norman; Auwerx, Johan; Willemin, Gilles; Shah, Hardik; Hartil, Kirsten; Vaitheesvaran, Bhavapriya; Kurland, Irwin; Hernandez, Nouria; Willis, Ian M

    2015-05-01

    MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences. PMID:25934505

  14. Inhibition of Wilms tumor 1 transactivation by bone marrow zinc finger 2, a novel transcriptional repressor.

    PubMed

    Lee, Tae Ho; Lwu, Shelly; Kim, Jungho; Pelletier, Jerry

    2002-11-22

    The Wilms tumor suppressor gene, wt1, encodes a zinc finger transcription factor that has been implicated in the regulation of a number of genes. Protein-protein interactions are known to modulate the transcription regulatory functions of Wilms tumor (WT1) and have also implicated WT1 in splicing. In this report, we identify a novel WT1-interacting protein, bone marrow zinc finger 2 (BMZF2), by affinity chromatography utilizing immobilized WT1 protein. BMZF2 is a potential transcription factor with 18 zinc fingers. The BMZF2 mRNA is mainly expressed in fetal tissues, and the protein is predominantly nuclear. Co-immunoprecipitation experiments are consistent with an in vivo association between WT1 and BMZF2. Glutathione S-transferase pulldown assays and far Western blots revealed that zinc fingers VI-X (amino acids 231-370) are required for interaction with the zinc finger region of WT1. Functionally, BMZF2 inhibits transcriptional activation by WT1. Moreover, a chimeric protein generated by fusion of BMZF2 to the GAL4 DNA-binding domain significantly decreases promoter activity of a reporter containing GAL4 DNA-binding sites, suggesting the presence of an active repressor domain within BMZF2. Our results suggest that BMZF2 interferes with the transactivation potential of WT1. PMID:12239212

  15. Transcription repressor activity of spleen tyrosine kinase mediates breast tumor suppression

    PubMed Central

    Wang, Lei; Devarajan, Eswaran; He, Jin; Reddy, Sekhar P.; Le Dai, Jia

    2005-01-01

    Spleen tyrosine kinase (SYK) is a candidate tumor suppressor gene in breast. Loss of SYK expression in breast tumors as a result of DNA hypermethylation promotes tumor cell proliferation and invasion and predicts shorter survival of breast cancer patients. We previously reported that, in addition to its well-known cytoplasmic localization, the full length Syk is also present in the nucleus, and that Syk nuclear translocation is a rate-limiting step to determine Syk tumor suppressor function. Here we show that the full-length form of Syk acts as a transcription repressor in the cell nucleus. Ectopic expression of Syk down-regulates the transcription of FRA1 and cyclin D1 oncogenes. This transcription repressing activity of Syk is associated with its binding to members of the histone deacetylase family. Syk interacts with transcription factor Sp1 at the Sp1 DNA-binding site in the FRA1 promoter to repress Sp1-activated FRA1 transcription. Thus, breast tumorigenesis and progression resulting from the loss of SYK are underscored by the de-repression of Sp1-mediated oncogene transcription. PMID:16288017

  16. Transcriptional repressor HipB regulates the multiple promoters in Escherichia coli.

    PubMed

    Lin, Chun-Yi; Awano, Naoki; Masuda, Hisako; Park, Jung-Ho; Inouye, Masayori

    2013-01-01

    HipB is a DNA-binding protein in Escherichia coli and negatively regulates its own promoter by binding to the palindromic sequences [TATCCN8GGATA (N represents any nucleotides)] on the hipBA promoter. For such sequences, bioinformatic analysis revealed that there are a total of 39 palindromic sequences (TATCCN(x)GGATA: N is any nucleotides and x is the number of nucleotides from 1 to 30) in the promoter regions of 33 genes on the E. coli genome. Notably, eutH and fadH have two and three TATCCN(x)GGATA palindromic sequences located in their promoters, respectively. Another significant finding was that a palindromic sequence was also identified in the promoter region of hipAB locus, known to be involved in the RelA-dependent persister cell formation in bacteria. Here, we demonstrated that HipB binds to the palindromic structures in the eutH, fadH, as well as the relA promoter regions and represses their expressions. We further demonstrated that HipA enhances the repression of the relA promoter activity by HipB. This effect was not observed with D291A HipA mutant which was previously shown to lack an ability to interact with HipB, indicating that HipA enhances the HipB's repressor activity through direct interaction with HipB.

  17. DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation

    PubMed Central

    Boedicker, James Q.; Garcia, Hernan G.; Johnson, Stephanie; Phillips, Rob

    2014-01-01

    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. PMID:24231252

  18. 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. PMID:27503074

  19. 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

  20. The CreA repressor is the sole DNA-binding protein responsible for carbon catabolite repression of the alcA gene in Aspergillus nidulans via its binding to a couple of specific sites.

    PubMed

    Panozzo, C; Cornillot, E; Felenbok, B

    1998-03-13

    Carbon catabolite repression is mediated in Aspergillus nidulans by the negative acting protein CreA. The CreA repressor plays a major role in the control of the expression of the alc regulon, encoding proteins required for the ethanol utilization pathway. It represses directly, at the transcriptional level, the specific transacting gene alcR, the two structural genes alcA and aldA, and other alc genes in all physiological growth conditions. Among the seven putative CreA sites identified in the alcA promoter region, we have determined the CreA functional targets in AlcR constitutive and derepressed genetic backgrounds. Two different divergent CreA sites, of which one overlaps a functional AlcR inverted repeat site, are largely responsible for alcA repression. Totally derepressed alcA expression is achieved when these two CreA sites are disrupted in addition to another single site, which overlaps the functional palindromic induction target. The fact that derepression is always associated with alcA overexpression is consistent with a competition model between AlcR and CreA for their cognate targets in the same region of the alcA promoter. Our results also indicate that the CreA repressor is necessary and sufficient for the total repression of the alcA gene.

  1. The Transcription Repressor REST in Adult Neurons: Physiology, Pathology, and Diseases1,2,3

    PubMed Central

    Baldelli, Pietro

    2015-01-01

    Abstract REST [RE1-silencing transcription factor (also called neuron-restrictive silencer factor)] is known to repress thousands of possible target genes, many of which are neuron specific. To date, REST repression has been investigated mostly in stem cells and differentiating neurons. Current evidence demonstrates its importance in adult neurons as well. Low levels of REST, which are acquired during differentiation, govern the expression of specific neuronal phenotypes. REST-dependent genes encode important targets, including transcription factors, transmitter release proteins, voltage-dependent and receptor channels, and signaling proteins. Additional neuronal properties depend on miRNAs expressed reciprocally to REST and on specific splicing factors. In adult neurons, REST levels are not always low. Increases occur during aging in healthy humans. Moreover, extensive evidence demonstrates that prolonged stimulation with various agents induces REST increases, which are associated with the repression of neuron-specific genes with appropriate, intermediate REST binding affinity. Whether neuronal increases in REST are protective or detrimental remains a subject of debate. Examples of CA1 hippocampal neuron protection upon depolarization, and of neurodegeneration upon glutamate treatment and hypoxia have been reported. REST participation in psychiatric and neurological diseases has been shown, especially in Alzheimer’s disease and Huntington’s disease, as well as epilepsy. Distinct, complex roles of the repressor in these different diseases have emerged. In conclusion, REST is certainly very important in a large number of conditions. We suggest that the conflicting results reported for the role of REST in physiology, pathology, and disease depend on its complex, direct, and indirect actions on many gene targets and on the diverse approaches used during the investigations. PMID:26465007

  2. Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers.

    PubMed

    Philibert, Robert A; Beach, Steven R H; Brody, Gene H

    2012-11-01

    Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10 (-7) and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure.

  3. Flexibility in the inducer binding region is crucial for allostery in the Escherichia coli lactose repressor.

    PubMed

    Xu, Jia; Matthews, Kathleen S

    2009-06-01

    Lactose repressor protein (LacI) utilizes an allosteric mechanism to regulate transcription in Escherichia coli, and the transition between inducer- and operator-bound states has been simulated by targeted molecular dynamics (TMD). The side chains of amino acids 149 and 193 interact and were predicted by TMD simulation to play a critical role in the early stages of the LacI conformational change. D149 contacts IPTG directly, and variations at this site provide the opportunity to dissect its role in inducer binding and signal transduction. Single mutants at D149 or S193 exhibit a minimal change in operator binding, and alterations in inducer binding parallel changes in operator release, indicating normal allosteric response. The observation that the double mutant D149A/S193A exhibits wild-type properties excludes the requirement for inter-residue hydrogen bond formation in the allosteric response. The double mutant D149C/S193C purified from cell extracts shows decreased sensitivity to inducer binding while retaining wild-type binding affinities and kinetic constants for both operator and inducer. By manipulating cysteine oxidation, we show that the more reduced state of D149C/S193C responds to inducer more like the wild-type protein, whereas the more oxidized state displays diminished inducer sensitivity. These features of D149C/S193C indicate that the novel disulfide bond formed in this mutant impedes the allosteric transition, consistent with the role of this region predicted by TMD simulation. Together, these results establish the requirement for flexibility in the spatial relationship between D149 and S193 rather than a specific D149-S193 interaction in the LacI allosteric response to inducer. PMID:19368358

  4. Transcriptional repressor Tbx3 is required for the hormone-sensing cell lineage in mammary epithelium.

    PubMed

    Kunasegaran, Kamini; Ho, Victor; Chang, Ted H-T; De Silva, Duvini; Bakker, Martijn L; Christoffels, Vincent M; Pietersen, Alexandra M

    2014-01-01

    The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development) and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production). Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER) and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells.

  5. Dissection of the PHO pathway in Schizosaccharomyces pombe using epistasis and the alternate repressor adenine.

    PubMed

    Estill, Molly; Kerwin-Iosue, Christine L; Wykoff, Dennis D

    2015-05-01

    In Saccharomyces cerevisiae, intracellular phosphate levels are maintained by the PHO pathway, activation of which is assayed by increased phosphatase activity. The PHO pathway of Schizosaccharomyces pombe upregulates phosphatase activity (encoded by pho1 (+)) during low extracellular phosphate levels, but the underlying mechanism is poorly understood. We utilized an alternate repressor of pho1 (+) expression (adenine supplementation) along with epistasis analysis to develop a model of how S. pombe PHO pathway components interact. Analyzing Pho1 activity in S. pombe PHO pathway deletion mutants during adenine starvation, we observed most mutants with a phosphatase defect in phosphate starvation also had a defect in adenine starvation. Pho7, a transcription factor in the PHO pathway, is necessary for an adenine starvation-mediated increase in Pho1 activity. Comparing adenine starvation to phosphate starvation, there are differences in the degree to which individual mutants regulate the two responses. Through epistasis studies, we identified two positive regulatory arms and one repressive arm of the PHO pathway. PKA activation is a positive regulator of Pho1 activity under both environmental conditions and is critical for transducing adenine concentrations in the cell. The synthesis of IP7 also appears critical for the induction of Pho1 activity during adenine starvation, but IP7 is not critical during phosphate starvation, which differs from S. cerevisiae. Finally, Csk1 is critical for repression of pho1 (+) expression during phosphate starvation. We believe all of these regulatory arms converge to increase transcription of pho1 (+) and some of the regulation acts through pho7 (+).

  6. JAZ Repressors: Potential Involvement in Nutrients Deficiency Response in Rice and Chickpea

    PubMed Central

    Singh, Ajit P.; Pandey, Bipin K.; Deveshwar, Priyanka; Narnoliya, Laxmi; Parida, Swarup K.; Giri, Jitender

    2015-01-01

    Jasmonates (JA) are well-known phytohormones which play important roles in plant development and defense against pathogens. Jasmonate ZIM domain (JAZ) proteins are plant-specific proteins and act as transcriptional repressors of JA-responsive genes. JA regulates both biotic and abiotic stress responses in plants; however, its role in nutrient deficiency responses is very elusive. Although, JA is well-known for root growth inhibition, little is known about behavior of JAZ genes in response to nutrient deficiencies, under which root architectural alteration is an important adaptation. Using protein sequence homology and a conserved-domains approach, here we identify 10 novel JAZ genes from the recently sequenced Chickpea genome, which is one of the most nutrient efficient crops. Both rice and chickpea JAZ genes express in tissue- and stimuli-specific manners. Many of which are preferentially expressed in root. Our analysis further showed differential expression of JAZ genes under macro (NPK) and micronutrients (Zn, Fe) deficiency in rice and chickpea roots. While both rice and chickpea JAZ genes showed a certain level of specificity toward type of nutrient deficiency, generally majority of them showed induction under K deficiency. Generally, JAZ genes showed an induction at early stages of stress and expression declined at later stages of macro-nutrient deficiency. Our results suggest that JAZ genes might play a role in early nutrient deficiency response both in monocot and dicot roots, and information generated here can be further used for understanding the possible roles of JA in root architectural alterations for nutrient deficiency adaptations. PMID:26617618

  7. SV40 early-to-late switch involves titration of cellular transcriptional repressors.

    PubMed

    Wiley, S R; Kraus, R J; Zuo, F; Murray, E E; Loritz, K; Mertz, J E

    1993-11-01

    We have purified factors from HeLa cell nuclear extracts that bind to the transcriptional initiation site of the SV40 major late promoter (SV40-MLP). The resulting fraction consists predominantly of three proteins, collectively called initiator-binding protein of SV40 (IBP-s) with electrophoretic mobilities of approximately 45-55 kD. Gel mobility-shift and DNase I-protection analyses indicate that each of these three proteins associates with high affinity to sequences located at the initiation site and 55 bp downstream of it. IBP-s-binding sites with lower affinities are located at +5 and +30. Addition of purified IBP-s to a cell-free transcription system represses transcription from the SV40-MLP, but not the SV40 early promoter. SV40 mutants lacking the two strongest IBP-s-binding sites (1) are not repressed by the addition of IBP-s in vitro, (2) overproduce late RNA (relative to wild-type SV40) at low, but not high, template copy number in vitro, and (3) exhibit increased levels of late RNA at early, but not late, times after transfection into CV-1 cells. Therefore, IBP-s is a cellular repressor of transcription of the SV40-MLP that may, in large part, be responsible for the replication-dependent component of the early-to-late shift in SV40 gene expression. Partial amino acid sequence data obtained from the approximately 55-kD component of IBP-s indicate that it is hERR1, an orphan member of the steroid-thyroid hormone receptor superfamily. These findings suggest simple molecular mechanisms by which hormones may modulate expression of viral late genes. We speculate that activation of expression of the late genes of other viruses may occur by similar mechanisms.

  8. 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 suprabasal cells were observed. E8 mt genomes were amplified to higher copy numbers and showed increased L1 transcripts compared to wt genomes. Furthermore, the number of cells expressing the viral late protein E4 or L1 or amplifying viral genomes was greatly increased in E8 mt cell lines. In wild-type cells, E8^E2C transcript levels did not decrease by differentiation. Our data indicate that the E8^E2C repressor limits viral transcription and replication throughout the complete life cycle of HPV16. PMID:24198405

  9. Differential Expression of Glucocorticoid Receptor Noncoding RNA Repressor Gas5 in Autoimmune and Inflammatory Diseases.

    PubMed

    Mayama, T; Marr, A K; Kino, T

    2016-08-01

    Glucocorticoids have strong regulatory actions on the immune system and act as potent therapeutic compounds for autoimmune and inflammatory diseases. We previously reported that the long noncoding RNA growth arrest-specific 5 (Gas5), which accumulates inside the cells in response to cellular starvation/growth arrest, functions as a potent repressor of the glucocorticoid receptor (GR) through its RNA "glucocorticoid response element (GRE)". To evaluate potential roles of Gas5 in immune-related disorders, we examined Gas5 RNA levels in various autoimmune, inflammatory, and infectious diseases using the microarray data available in the Gene Expression Omnibus. We found that Gas5 levels were altered in whole blood or leukocytes of the patients with rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and sarcoidosis. Gas5 levels were also altered in infectious diseases, such as by the human immunodeficiency virus type-1 and influenza virus, and bacterial sepsis. In our experimental analysis using mice, Gas5 levels were kept at high basal levels and did not respond to fasting in immune organs, such as spleen and thymus, while its levels in metabolic organs, including liver, fat, and skeletal muscles, were low at baseline and were highly elevated upon this treatment, possibly through suppression of the mTOR pathway. These results suggest that Gas5 plays a role in the regulation of immune functions and pathogenesis/pathophysiology of autoimmune, inflammatory, and infectious diseases in part through modulation of the GR transcriptional activity via its decoy RNA "GRE". Changes in the Gas5 levels may also influence disease response to immunosuppressive glucocorticoid therapy. PMID:27214311

  10. 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

  11. Investigation of Changes in Tetracycline Repressor Binding upon Mutations in the Tetracycline Operator.

    PubMed

    Bolintineanu, Dan S; Volzing, Katherine; Vivcharuk, Victor; Sayyed-Ahmad, Abdallah; Srivastava, Poonam; Kaznessis, Yiannis N

    2014-10-01

    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.

  12. DNA Methylation of the Aryl Hydrocarbon Receptor Repressor Associations with Cigarette Smoking and Subclinical Atherosclerosis

    PubMed Central

    Reynolds, Lindsay M.; Wan, Ma; Ding, Jingzhong; Taylor, Jackson R.; Mstat, Kurt Lohman; Su, Dan; Bennett, Brian D.; Porter, Devin K.; Gimple, Ryan; Pittman, Gary S.; Wang, Xuting; Howard, Timothy D.; Siscovick, David; Psaty, Bruce M.; Shea, Steven; Burke, Gregory L.; Jacobs, David R.; Rich, Stephen S.; Hixson, James E.; Stein, James H.; Stunnenberg, Hendrik; Barr, R. Graham; Kaufman, Joel D.; Post, Wendy S.; Hoeschele, Ina; Herrington, David M.; Bell, Douglas A.; Liu, Yongmei

    2015-01-01

    Background Tobacco smoke contains numerous agonists of the aryl-hydrocarbon receptor (AhR) pathway, and activation of the AhR pathway was shown to promote atherosclerosis in mice. Intriguingly, cigarette smoking is most strongly and robustly associated with DNA modifications to an AhR pathway gene, the aryl-hydrocarbon receptor repressor (AHRR). We hypothesized that altered AHRR methylation in monocytes, a cell type sensitive to cigarette smoking and involved in atherogenesis, may be a part of the biological link between cigarette smoking and atherosclerosis. Methods and Results DNA methylation profiles of AHRR in monocytes (542 CpG sites ± 150kb of AHRR, using Illumina 450K array) were integrated with smoking habits and ultrasound-measured carotid plaque scores from 1,256 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Methylation of cg05575921 significantly associated (p = 6.1×10−134) with smoking status (current vs. never). Novel associations between cg05575921 methylation and carotid plaque scores (p = 3.1×10−10) were identified, which remained significant in current and former smokers even after adjusting for self-reported smoking habits, urinary cotinine, and well-known CVD risk factors. This association replicated in an independent cohort using hepatic DNA (n = 141). Functionally, cg05575921 was located in a predicted gene expression regulatory element (enhancer), and had methylation correlated with AHRR mRNA profiles (p = 1.4×10−17) obtained from RNA sequencing conducted on a subset (n = 373) of the samples. Conclusions These findings suggest AHRR methylation may be functionally related to AHRR expression in monocytes, and represents a potential biomarker of subclinical atherosclerosis in smokers. PMID:26307030

  13. Transcriptional Repressor Tbx3 Is Required for the Hormone-Sensing Cell Lineage in Mammary Epithelium

    PubMed Central

    Kunasegaran, Kamini; Ho, Victor; Chang, Ted H-. T.; De Silva, Duvini; Bakker, Martijn L.; Christoffels, Vincent M.; Pietersen, Alexandra M.

    2014-01-01

    The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development) and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production). Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER) and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells. PMID:25343378

  14. 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.

  15. Nkx1-2 is a transcriptional repressor and is essential for the activation of Brachyury in P19 mouse embryonal carcinoma cell

    PubMed Central

    Tamashiro, Dana Ann A.; Alarcon, Vernadeth B.; Marikawa, Yusuke

    2013-01-01

    Activation of Wnt/β-catenin signaling is crucial for the differentiation of pluripotent stem cells, namely the epiblast, embryonic stem, and embryonal carcinoma cells, into mesendoderm. However, downstream events of Wnt/β-catenin signaling that control the formation of mesendoderm are still unclear. In the present study, we used mouse P19 embryonal carcinoma cells as a model, and identified a homeodomain protein Nkx1-2 as a key regulator of mesendoderm formation. In the mouse embryo, Nkx1-2 was expressed in the primitive streak, in which the nascent mesendoderm emerges. In P19 cells, the expression of Nkx1-2 was activated by Wnt/β-catenin signaling independently of Brachyury, an evolutionary conserved early mesendoderm gene. In contrast, the expression of Nkx1-2 was both necessary and sufficient for the activation of Brachyury. Nkx1-2 acted as a transcriptional repressor to mediate the action of Wnt/β-catenin signaling to activate the Brachyury expression. We found Tcf3 as a potential target of gene repression by Nkx1-2, and the down-regulation of Tcf3 was partly required for effective activation of Brachyury by Wnt/β-catenin signaling. These results suggest that Nkx1-2 is a critical component of the gene regulatory network that operates downstream of Wnt/β-catenin signaling to regulate the formation of mesendoderm. PMID:22475651

  16. The banana fruit Dof transcription factor MaDof23 acts as a repressor and interacts with MaERF9 in regulating ripening-related genes

    PubMed Central

    Feng, Bi-hong; Han, Yan-chao; Xiao, Yun-yi; Kuang, Jian-fei; Fan, Zhong-qi; Chen, Jian-ye; Lu, Wang-jin

    2016-01-01

    The DNA binding with one finger (Dof) proteins, a family of plant-specific transcription factors, are involved in a variety of plant biological processes. However, little information is available on their involvement in fruit ripening. We have characterized 25 MaDof genes from banana fruit (Musa acuminata), designated as MaDof1–MaDof25. Gene expression analysis in fruit subjected to different ripening conditions revealed that MaDofs were differentially expressed during different stages of ripening. MaDof10, 23, 24, and 25 were ethylene-inducible and nuclear-localized, and their transcript levels increased during fruit ripening. Moreover, yeast two-hybrid and bimolecular fluorescence complementation analyses demonstrated a physical interaction between MaDof23 and MaERF9, a potential regulator of fruit ripening reported in a previous study. We determined that MaDof23 is a transcriptional repressor, whereas MaERF9 is a transcriptional activator. We suggest that they might act antagonistically in regulating 10 ripening-related genes, including MaEXP1/2/3/5, MaXET7, MaPG1, MaPME3, MaPL2, MaCAT, and MaPDC, which are associated with cell wall degradation and aroma formation. Taken together, our findings provide new insight into the transcriptional regulation network controlling banana fruit ripening. PMID:26889012

  17. Germ cell expression of the transcriptional co-repressor TIF1beta is required for the maintenance of spermatogenesis in the mouse.

    PubMed

    Weber, Philipp; Cammas, Florence; Gerard, Christelle; Metzger, Daniel; Chambon, Pierre; Losson, Régine; Mark, Manuel

    2002-05-01

    The gene for transcriptional intermediary factor 1beta (TIF1beta) encodes a transcriptional co-repressor known to play essential roles in chromatin remodeling as well as in early embryonic development. During spermatogenesis, TIF1beta is preferentially associated with heterochromatin structures of Sertoli cells and round spermatids, as well as with meiotic chromosomes. Its expression is tightly regulated within spermatocyte and spermatid populations, and it is undetectable in spermatogonia. Spatiotemporally controlled ablation of TIF1beta by using a germ cell lineage-specific CreER(T)/loxP system leads to testicular degeneration. This degeneration is not due to impairment of chromatin remodeling processes during meiosis and spermiogenesis, as TIF1beta-deficient spermatocytes are able to complete their differentiation into spermatozoa. It rather occurs as a consequence of shedding of immature germ cells (spermatocytes and spermatids), and disappearance of stem spermatogonia. These results indicate that TIF1beta has important functions in the homeostasis of the seminiferous epithelium, and probably plays a crucial role in the network of paracrine interactions between germ cell subpopulations and/or Sertoli cells. PMID:11973266

  18. Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system.

    PubMed Central

    Winstanley, C; Morgan, J A; Pickup, R W; Jones, J G; Saunders, J R

    1989-01-01

    Plasmid systems with unique markers were constructed to assess the fate of recombinant DNA and genetically manipulated bacteria in soil and freshwater model environments. On such constructs the marker gene, xylE (for catechol 2,3-dioxygenase), is expressed from the lambda promoter pL or pR, each of which is controlled by the temperature-sensitive lambda repressor c1857. Combinations of these elements were cloned into the broad-host-range plasmid pKT230 to form pLV1010 (pL-xylE), pLV1011 (pL-xylE-c1857), and pLV1013 (pR-xylE-c1857). The recombinant plasmids were introduced into different gram-negative bacteria. The thermoregulated system of pLV1013 functioned well in a range of species, with xylE induction being readily achieved by elevation of the temperature from 28 to 37 degrees C. There was a difference in the induction of catechol 2,3-dioxygenase activity, depending on whether xylE was expressed from pL (pLV1011) or pR (pLV1013). Our observations on testing the different systems in a number of hosts suggest that genes carried by the DNA of genetically engineered microorganisms may not be expressed in a predictable manner following transfer from the release host to other species. Images PMID:2729979

  19. Hes-1, a known transcriptional repressor, acts as a transcriptional activator for the human acid alpha-glucosidase gene in human fibroblast cells.

    PubMed

    Yan, Bo; Raben, Nina; Plotz, Paul H

    2002-03-01

    Hes-1, the mammalian homologue 1 of Drosophila hairy and Enhancer of split proteins, belongs to a family of basic helix-loop-helix proteins that are essential to neurogenesis, myogenesis, hematopoiesis, and sex determination. Hes-1 is a transcriptional repressor for a number of known genes including the human acid alpha-glucosidase (GAA) gene as we have previously shown in Hep G2 cells. The human GAA gene encodes the enzyme for glycogen breakdown in lysosomes, deficiency of which results in Glycogen Storage Disease type II (Pompe syndrome). Using constructs containing the DNA element that demonstrates repressive activity in Hep G2 cells and conditions in which the same transcription factors, Hes-1 and YY1, bind, we have shown that this element functions as an enhancer in human fibroblasts. Site-directed mutagenesis and overexpression of Hes-1 showed that Hes-1 functions as a transcriptional activator. The dual function of Hes-1 we have found is likely to contribute to the subtle tissue-specific control of this housekeeping gene.

  20. The banana fruit Dof transcription factor MaDof23 acts as a repressor and interacts with MaERF9 in regulating ripening-related genes.

    PubMed

    Feng, Bi-hong; Han, Yan-chao; Xiao, Yun-yi; Kuang, Jian-fei; Fan, Zhong-qi; Chen, Jian-ye; Lu, Wang-jin

    2016-04-01

    The DNA binding with one finger (Dof) proteins, a family of plant-specific transcription factors, are involved in a variety of plant biological processes. However, little information is available on their involvement in fruit ripening. We have characterized 25 MaDof genes from banana fruit (Musa acuminata), designated as MaDof1-MaDof25 Gene expression analysis in fruit subjected to different ripening conditions revealed that MaDofs were differentially expressed during different stages of ripening. MaDof10, 23, 24, and 25 were ethylene-inducible and nuclear-localized, and their transcript levels increased during fruit ripening. Moreover, yeast two-hybrid and bimolecular fluorescence complementation analyses demonstrated a physical interaction between MaDof23 and MaERF9, a potential regulator of fruit ripening reported in a previous study. We determined that MaDof23 is a transcriptional repressor, whereas MaERF9 is a transcriptional activator. We suggest that they might act antagonistically in regulating 10 ripening-related genes, including MaEXP1/2/3/5, MaXET7, MaPG1, MaPME3, MaPL2, MaCAT, and MaPDC, which are associated with cell wall degradation and aroma formation. Taken together, our findings provide new insight into the transcriptional regulation network controlling banana fruit ripening. PMID:26889012

  1. Understanding Mechanisms of GLI-Mediated Transcription during Craniofacial Development and Disease Using the Ciliopathic Mutant, talpid2

    PubMed Central

    Chang, Ya-Ting; Chaturvedi, Praneet; Schock, Elizabeth N.; Brugmann, Samantha A.

    2016-01-01

    The primary cilium is a ubiquitous, microtubule-based organelle that cells utilize to transduce molecular signals. Ciliopathies are a group of diseases that are caused by a disruption in the structure or function of the primary cilium. Over 30% of all ciliopathies are primarily defined by their craniofacial phenotypes, which typically include midfacial defects, cleft lip/palate, micrognathia, aglossia, and craniosynostosis. The frequency and severity of craniofacial phenotypes in ciliopathies emphasizes the importance of the cilium during development of the craniofacial complex. Molecularly, many ciliopathic mutants, including the avian talpid2 (ta2), report pathologically high levels of full-length GLI3 (GLI3FL), which can go on to function as an activator (GLIA), and reduced production of truncated GLI3 (GLI3T), which can go on to function as a repressor (GLIR). These observations suggest that the craniofacial phenotypes of ciliary mutants like ta2 are caused either by excessive activity of the GLIA or reduced activity of GLIR. To decipher between these two scenarios, we examined GLI3 occupation at the regulatory regions of target genes and subsequent target gene expression. Using in silico strategies we identified consensus GLI binding regions (GBRs) in the avian genome and confirmed GLI3 binding to the regulatory regions of its targets by chromatin immunoprecipitation (ChIP). In ta2 mutants, there was a strikingly low number of GLI3 target genes that had significantly increased expression in facial prominences compared to the control embryo and GLI3 occupancy at GBRs associated with target genes was largely reduced. In vitro DNA binding assays, further supported ChIP results, indicated that the excessive GLI3FL generated in ta2 mutants did not bind to GBRs. In light of these results, we explored the possibility of GLI co-regulator proteins playing a role in regulatory mechanism of GLI-mediated transcription. Taken together our studies suggest that craniofacial

  2. Escherichia coli purine repressor: key residues for the allosteric transition between active and inactive conformations and for interdomain signaling.

    PubMed

    Lu, F; Brennan, R G; Zalkin, H

    1998-11-10

    The Escherichia coli purine repressor, PurR, exists in an equilibrium between open and closed conformations. Binding of a corepressor, hypoxanthine or guanine, shifts the allosteric equilibrium in favor of the closed conformation and increases the operator DNA binding affinity by 40-fold compared to aporepressor. Glu70 and Trp147 PurR mutations were isolated which perturb the allosteric equilibrium. Three lines of evidence indicate that the allosteric equilibrium of E70A and W147A aporepressors was shifted toward the closed conformation. First, compared to wild-type PurR, these mutant repressors had a 10-30-fold higher corepressor binding affinity. Second, the mutant aporepressors bound to operator DNA with an affinity that is characteristic of the wild-type PurR holorepressor. Third, binding of guanine to wild-type PurR resulted in a near-UV circular dichroism spectral change at 297-305 nm that is attributed to the closed conformation. The circular dichroism spectrum of the E70A aporepressor at 297-305 nm was that expected for the closed conformation, and it was not appreciably altered by corepressor binding. Mutational analysis was used to identify an Arg115-Ser46' interdomain intersubunit hydrogen bond that is necessary for transmitting the allosteric transition in the corepressor binding domain to the DNA binding domain. R115A and S46G PurR mutants were defective in DNA binding in vitro and repressor function in vivo although corepressor binding was identical to the wild type. These results establish that the hydrogen bond between the side chain NH2 of Arg115 and the main chain CO of Ser46' plays a critical role in interdomain signaling.

  3. Lac Repressor Mediated DNA Looping: Monte Carlo Simulation of Constrained DNA Molecules Complemented with Current Experimental Results

    PubMed Central

    Biton, Yoav Y.; Kumar, Sandip; Dunlap, David; Swigon, David

    2014-01-01

    Tethered particle motion (TPM) experiments can be used to detect time-resolved loop formation in a single DNA molecule by measuring changes in the length of a DNA tether. Interpretation of such experiments is greatly aided by computer simulations of DNA looping which allow one to analyze the structure of the looped DNA and estimate DNA-protein binding constants specific for the loop formation process. We here present a new Monte Carlo scheme for accurate simulation of DNA configurations subject to geometric constraints and apply this method to Lac repressor mediated DNA looping, comparing the simulation results with new experimental data obtained by the TPM technique. Our simulations, taking into account the details of attachment of DNA ends and fluctuations of the looped subsegment of the DNA, reveal the origin of the double-peaked distribution of RMS values observed by TPM experiments by showing that the average RMS value for anti-parallel loop types is smaller than that of parallel loop types. The simulations also reveal that the looping probabilities for the anti-parallel loop types are significantly higher than those of the parallel loop types, even for loops of length 600 and 900 base pairs, and that the correct proportion between the heights of the peaks in the distribution can only be attained when loops with flexible Lac repressor conformation are taken into account. Comparison of the in silico and in vitro results yields estimates for the dissociation constants characterizing the binding affinity between O1 and Oid DNA operators and the dimeric arms of the Lac repressor. PMID:24800809

  4. Insight into a Physiological Role for the EC Night-Time Repressor in the Arabidopsis Circadian Clock.

    PubMed

    Mizuno, Takeshi; Kitayama, Miki; Takayama, Chieko; Yamashino, Takafumi

    2015-09-01

    Life cycle adaptation to seasonal variation in photoperiod and temperature is a major determinant of ecological success of widespread domestication of Arabidopsis thaliana. The circadian clock plays a role in the underlying mechanism for adaptation. Nevertheless, the mechanism by which the circadian clock tracks seasonal changes in photoperiod and temperature is a longstanding subject of research in the field. We previously showed that a set of the target genes (i.e. GI, LNK1. PRR9 and PRR7) of the Evening Complex (EC) consisting of LUX-ELF3-ELF4 is synergistically induced in response to both warm-night and night-light signals. Here, we further show that the responses occur within a wide range of growth-compatible temperatures (16-28°C) in response to a small change in temperature (Δ4°C). A dim light pulse (<1 µmol m(-2) s(-1)) causes the enhanced effect on the transcription of EC targets. The night-light pulse antagonizes against a positive effect of the cool-night signal on the EC activity. The mechanism of double-checking external temperature and light signals through the EC nighttime repressor might enable plants to ignore (or tolerate) daily fluctuation of ambient temperature within a short time interval in their natural habitats. Taken together, the EC night-time repressor might play a physiological role in tracking seasonal variation in photoperiod and temperature by conservatively double-checking both the light and temperature conditions. Another EC target output gene PIF4 regulating plant morphologies is also regulated by both the temperature and light stimuli during the night. Hence, the EC night-time repressor is also implicated in a physiological output of the PIF4-mediated regulation of morphologies in response to seasonal variation in photoperiod and ambient temperature.

  5. Ectopic expression and knockdown of a zebrafish sox21 reveal its role as a transcriptional repressor in early development.

    PubMed

    Argenton, Francesco; Giudici, Simona; Deflorian, Gianluca; Cimbro, Simona; Cotelli, Franco; Beltrame, Monica

    2004-02-01

    Sox proteins are DNA-binding proteins belonging to the HMG box superfamily and they play key roles in animal embryonic development. Zebrafish Sox21a is part of group B Sox proteins and its chicken and mouse orthologs have been described as transcriptional repressor and activator, respectively, in two different target gene contexts. Zebrafish sox21a is present as a maternal transcript in the oocyte and is mainly expressed at the developing midbrain-hindbrain boundary from the onset of neurulation. In order to understand its role in vivo, we ectopically expressed sox21a by microinjection. Ectopic expression of full length sox21a leads to dorsalization of the embryos. A subset of the dorsalized embryos shows a partial axis splitting, and hence an ectopic neural tube, as an additional phenotype. At gastrulation, injected embryos show expansion of the expression domains of organizer-specific genes, such as chordin and goosecoid. Molecular markers used in somitogenesis highlight that sox21a-injected embryos have shortened AP axis, undulating axial structures, enlarged or even radialized paraxial territory. The developmental abnormalities caused by ectopic expression of sox21a are suggestive of defects in convergence-extension morphogenetic movements. Antisense morpholino oligonucleotides, designed to functionally knockdown sox21a, cause ventralization of the embryos. Moreover, gain-of-function experiments with chimeric constructs, where Sox21a DNA-binding domain is fused to a transcriptional activator (VP16) or repressor (EnR) domain, suggests that zebrafish Sox21a acts as a repressor in dorso-ventral patterning.

  6. Identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse hepatocyte growth factor gene.

    PubMed Central

    Liu, Y; Beedle, A B; Lin, L; Bell, A W; Zarnegar, R

    1994-01-01

    Hepatocyte growth factor (HGF), a cytokine with multiple functions, exhibits cell-type-specific as well as cytokine- and steroid hormone-regulated expression. The HGF gene is known to be expressed predominately in mesenchymal but not in epithelial cells. In this study, we report the identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse HGF gene, which is evidently responsible for the suppression of HGF expression in epithelial cells. Gel mobility shift assays and DNase I footprinting studies revealed that a 27-bp element (-16 to +11) around the transcription initiation site is responsible for the binding of a nuclear protein which is present in epithelial but not in mesenchymally derived cells. Further analysis of the binding activity of the DNA region with nuclear protein revealed that an approximately 19-bp sequence containing a unique palindromic structure (5'-AACCGACCGGTT-3') overlapped by a CAP box is essential for binding. Substitution of a single base (the contact site) within this region by site-directed mutagenesis resulted in total abrogation of the binding of the nuclear protein and a concomitant increase in the transcriptional activity of various lengths of HGF-chloramphenicol acetyltransferase fused genes when transfected into the epithelial cell line RL95-2 but not the mesenchymal cell line NIH 3T3. Southwestern (DNA-protein) analyses revealed that the nuclear protein which binds to this repressor element is a single polypeptide of approximately 70 kDa. Analysis of the nuclear extract prepared from regenerating mouse liver at various times after two-thirds partial hepatectomy by gel mobility shift assay revealed a substantial reduction (more than 75% within 3 h) in the binding of the repressor to its cognate binding site. Our results suggest that a cis-acting transcriptional repressor in the promoter region of the mouse HGF gene is involved in cell-type-specific regulation through binding to its cognate

  7. Structural consequences of two methyl additions in the E. coli trp repressor L-tryptophan binding pocket

    SciTech Connect

    Lawson, C.L.

    1995-12-01

    The flexibility and specificity of the L-tryptophan corepressor binding pocket of E coli trp repressor are being investigated by high-resolution crystallographic examination of aporepressor/corepressor analog complexes. While addition of a methyl group on the corepressor indole (5-methyl-tryptophan) results in a small but measurable shift in the position of that functional group introduction of a methyl group on a nearby residue in the binding pocket (Val 58 {yields} Ile) leaves the indole position of L-tryptophan essentially unchanged. Careful alignment of these structures with aporepressor/L-tryptophan/operator-DNA complexes reveal why 5-methyltryptophan is a better corepressor than L-tryptophan.

  8. Probing the role of water in the tryptophan repressor-operator complex.

    PubMed

    Brown, M P; Grillo, A O; Boyer, M; Royer, C A

    1999-06-01

    The Escherichia coli tryptophan repressor protein (TR) represses the transcription of several genes in response to the concentration of tryptophan in the environment. In the co-crystal structure of TR bound to a DNA fragment containing its target very few direct contacts between TR and the DNA were observed. In contrast, a number of solvent mediated contacts were apparent. NMR solution structures, however, did not resolve any solvent mediated bonds at the complex interface. To probe for the role of water in TR operator recognition, the effect of osmolytes on the interactions between TR and a target oligonucleotide bearing the operator site was examined. In the absence of specific solvent mediated hydrogen bonding interactions between the protein and the DNA, increasing osmolyte concentration is expected to strongly stabilize the TR operator interaction due to the large amount of macromolecular surface area buried upon complexation. The results of our studies indicate that xylose did not alter the binding affinity significantly, while glycerol and PEG had a small stabilizing effect. A study of binding as a function of betaine concentration revealed that this osmolyte at low concentration results in a stabilization of the 1:1 TR/operator complex, but at higher concentrations leads to a switching between binding modes to favor tandem binding. Analysis of the effects of betaine on the 1:1 complex suggest that this osmolyte has about 78% of the expected effect. If one accepts the analysis in terms of the number of water molecules excluded upon complexation, these results suggest that about 75 water molecules remain at the interface of the 1:1 dimer/DNA complex. This value is consistent with the number of water molecules found at the interface in the crystallographically determined structure and supports the notion that interfacial waters play an important thermodynamic role in the specific complexation of one TR dimer with its target DNA. However, the complexity of the

  9. Conservation of the LexA repressor binding site in Deinococcus radiodurans.

    PubMed

    Khan, Feroz; Singh, S P; Mishra, B N

    2008-01-01

    The LexA protein is a transcriptional repressor of the bacterial SOS DNA repair system, which comprises a set of DNA repair and cellular survival genes that are induced in response to DNA damage. Its varied DNA binding motifs have been characterized and reported in the Escherichia coli, Bacillus subtilis, rhizobia family members, marine magnetotactic bacterium, Salmonella typhimurium and recently in Mycobacterium tuberculosis and this motifs information has been used in our theoretical analysis to detect its novel regulated genes in radio-resistant Deinococcus radiodurans genome. This bacterium showed presence of SOS-box like consensus sequence in the upstream sequences of 3166 genes with >60% motif score similarity percentage (MSSP) on both strands. Attempts to identify LexA-binding sites and the composition of the putative SOS regulon in D. radiodurans have been unsuccessful so far. To resolve the problem we performed theoretical analysis with modifications on reported data set of genes related to DNA repair (61 genes), stress response (145 genes) and some unusual predicted operons (21 clusters). Expression of some of the predicted SOS-box regulated operon members then was examined through the previously reported microarray data which confirm the expression of only single predicted operon i.e. DRB0143 (AAA superfamily NTPase related to 5-methylcytosine specific restriction enzyme subunit McrB) and DRB0144 (homolog of the McrC subunit of the McrBC restriction modification system). The methodology involved weight matrix construction through CONSENSUS algorithm using information of conserved upstream sequences of eight known genes including dinB, tagC, lexA, recA, uvrB, yneA of B. subtilis while lexA and recA of D. radiodurans through phylogenetic footprinting method and later detection of similar conserved SOS-box like LexA binding motifs through both RSAT & PoSSuMsearch programs. The resultant DNA consensus sequence had highly conserved 14 bp SOS-box like binding

  10. The Gfi-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal.

    PubMed Central

    Grimes, H L; Chan, T O; Zweidler-McKay, P A; Tong, B; Tsichlis, P N

    1996-01-01

    The Gfi-1 proto-oncogene is activated by provirus insertion in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced thymomas and encodes a nuclear, sequence-specific DNA-binding protein. Here we show that Gfi-1 is a position- and orientation-independent active transcriptional repressor, whose activity depends on a 20-amino-acid N-terminal repressor domain, coincident with a nuclear localization motif. The sequence of the Gfi-1 repressor domain is related to the sequence of the repressor domain of Gfi-1B, a Gfi-1-related protein, and to sequences at the N termini of the insulinoma-associated protein, IA-1, the homeobox protein Gsh-1, and the vertebrate but not the Drosophila members of the Snail-Slug protein family (Snail/Gfi-1, SNAG domain). Although not functionally characterized, these SNAG-related sequences are also likely to mediate transcriptional repression. Therefore, the Gfi-1 SNAG domain may be the prototype of a novel family of evolutionarily conserved repressor domains that operate in multiple cell lineages. Gfi-1 overexpression in IL-2-dependent T-cell lines allows the cells to escape from the G1 arrest induced by IL-2 withdrawal. Since a single point mutation in the SNAG domain (P2A) inhibits both the Gfi-1-mediated transcriptional repression and the G1 arrest induced by IL-2 starvation, we conclude that the latter depends on the repressor activity of the SNAG domain. Induction of Gfi-1 may therefore contribute to T-cell activation and tumor progression by repressing the expression of genes that inhibit cellular proliferation. PMID:8887656

  11. O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis

    PubMed Central

    Zentella, Rodolfo; Hu, Jianhong; Hsieh, Wen-Ping; Matsumoto, Peter A.; Dawdy, Andrew; Barnhill, Benjamin; Oldenhof, Harriëtte; Hartweck, Lynn M.; Maitra, Sushmit; Thomas, Stephen G.; Cockrell, Shelley; Boyce, Michael; Shabanowitz, Jeffrey; Hunt, Donald F.; Olszewski, Neil E.; Sun, Tai-ping

    2016-01-01

    The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein–protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors—PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)—that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development. PMID:26773002

  12. Thermodynamic and alkylation interference analysis of the lac repressor-operator substituted with the analogue 7-deazaguanine.

    PubMed

    Zhang, X; Gottlieb, P A

    1993-10-26

    Guanine residues in the lac operator were substituted with the isosteric analogue of 7-deazaguanine. The observed equilibrium dissociation constants for lac repressor binding to substituted operators were measured in 10 mM Tris, 150 mM KCl, 0.1 mM EDTA, and 0.1 mM DTE, pH 7.6, at 25 degrees C, using either a standard equilibrium assay or a competition assay. Of the seven individual sites tested, only three significantly altered the complex affinity. Two of these sites are symmetrically related and are positioned in the major grooves that are known to interact directly with repressor, and the third site is located in the central core region of the operator. The alkylation interference assay, which identifies essential phosphate sites, was used to define the extent of perturbation on the protein-DNA complex by the modified nucleotide. Chemical footprinting data for the singly substituted operator done at a single concentration of protein reflected the alignment of sterically incompatible groups or disruption in the local conformation, but did not appear to alter general phosphate backbone interactions. Similar experiments with a doubly substituted operator revealed that the free energy of binding was an additive combination of each of the individual sites, and the alkylation interference data were similar to the singly substituted and wild-type operators. A constitutive mutant which also had a higher binding constant had a similar alkylation interference pattern. PMID:8218202

  13. The Banana Transcriptional Repressor MaDEAR1 Negatively Regulates Cell Wall-Modifying Genes Involved in Fruit Ripening.

    PubMed

    Fan, Zhong-Qi; Kuang, Jian-Fei; Fu, Chang-Chun; Shan, Wei; Han, Yan-Chao; Xiao, Yun-Yi; Ye, Yu-Jie; Lu, Wang-Jin; Lakshmanan, Prakash; Duan, Xue-Wu; Chen, Jian-Ye

    2016-01-01

    Ethylene plays an essential role in many biological processes including fruit ripening via modulation of ethylene signaling pathway. Ethylene Response Factors (ERFs) are key transcription factors (TFs) involved in ethylene perception and are divided into AP2, RAV, ERF, and DREB sub-families. Although a number of studies have implicated the involvement of DREB sub-family genes in stress responses, little is known about their roles in fruit ripening. In this study, we identified a DREB TF with a EAR motif, designated as MaDEAR1, which is a nucleus-localized transcriptional repressor. Expression analysis indicated that MaDEAR1 expression was repressed by ethylene, with reduced levels of histone H3 and H4 acetylation at its regulatory regions during fruit ripening. In addition, MaDEAR1 promoter activity was also suppressed in response to ethylene treatment. More importantly, MaDEAR1 directly binds to the DRE/CRT motifs in promoters of several cell wall-modifying genes including MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 associated with fruit softening during ripening and represses their activities. These data suggest that MaDEAR1 acts as a transcriptional repressor of cell wall-modifying genes, and may be negatively involved in ethylene-mediated ripening of banana fruit. Our findings provide new insights into the involvement of DREB TFs in the regulation of fruit ripening.

  14. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

    PubMed

    Rizvi, Noreen F; Weaver, Jessica D; Cram, Erin J; Lee-Parsons, Carolyn W T

    2016-01-01

    The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis.

  15. The BRAF oncoprotein functions through the transcriptional repressor MAFG to mediate the CpG Island Methylator phenotype.

    PubMed

    Fang, Minggang; Ou, Jianhong; Hutchinson, Lloyd; Green, Michael R

    2014-09-18

    Most colorectal cancers (CRCs) containing activated BRAF (BRAF[V600E]) have a CpG island methylator phenotype (CIMP) characterized by aberrant hypermethylation of many genes, including the mismatch repair gene MLH1. MLH1 silencing results in microsatellite instability and a hypermutable phenotype. Through an RNAi screen, here we identify the transcriptional repressor MAFG as the pivotal factor required for MLH1 silencing and CIMP in CRCs containing BRAF(V600E). In BRAF-positive human CRC cell lines and tumors, MAFG is bound at the promoters of MLH1 and other CIMP genes, and recruits a corepressor complex that includes its heterodimeric partner BACH1, the chromatin remodeling factor CHD8, and the DNA methyltransferase DNMT3B, resulting in hypermethylation and transcriptional silencing. BRAF(V600E) increases BRAF/MEK/ERK signaling resulting in phosphorylation and elevated levels of MAFG, which drives DNA binding. Analysis of transcriptionally silenced CIMP genes in KRAS-positive CRCs indicates that different oncoproteins direct the assembly of distinct repressor complexes on common promoters.

  16. Transcriptional repressor domain of MBD1 is intrinsically disordered and interacts with its binding partners in a selective manner.

    PubMed

    Hameed, Umar Farook Shahul; Lim, Jackwee; Zhang, Qian; Wasik, Mariusz A; Yang, Daiwen; Swaminathan, Kunchithapadam

    2014-05-09

    Methylation of DNA CpG sites is a major mechanism of epigenetic gene silencing and plays important roles in cell division, development and carcinogenesis. One of its regulators is the 64-residue C-terminal Transcriptional Repressor Domain (the TRD) of MBD1, which recruits several repressor proteins such as MCAF1, HDAC3 and MPG that are essential for the gene silencing. Using NMR spectroscopy, we have characterized the solution structure of the C-terminus of MBD1 (MBD1-c, residues D507 to Q605), which included the TRD (A529 to P592). Surprisingly, the MBD1-c is intrinsically disordered. Despite its lack of a tertiary folding, MBD1-c could still bind to different partner proteins in a selective manner. MPG and MCAF1Δ8 showed binding to both the N-terminal and C-terminal residues of MBD1-c but HDAC3 preferably bound to the C-terminal region. This study reveals how MBD1-c discriminates different binding partners, and thus, expands our understanding of the mechanisms of gene regulation by MBD1.

  17. CD8 lineage-specific regulation of interleukin-7 receptor expression by the transcriptional repressor Gfi1.

    PubMed

    Ligons, Davinna L; Tuncer, Ceren; Linowes, Brett A; Akcay, Izzet Mehmet; Kurtulus, Sema; Deniz, Emre; Atasever Arslan, Belkis; Cevik, Safak Isil; Keller, Hilary R; Luckey, Megan A; Feigenbaum, Lionel; Möröy, Tarik; Ersahin, Tulin; Atalay, Rengul; Erman, Batu; Park, Jung-Hyun

    2012-10-01

    Interleukin-7 receptor α (IL-7Rα) is essential for T cell survival and differentiation. Glucocorticoids are potent enhancers of IL-7Rα expression with diverse roles in T cell biology. Here we identify the transcriptional repressor, growth factor independent-1 (Gfi1), as a novel intermediary in glucocorticoid-induced IL-7Rα up-regulation. We found Gfi1 to be a major inhibitory target of dexamethasone by microarray expression profiling of 3B4.15 T-hybridoma cells. Concordantly, retroviral transduction of Gfi1 significantly blunted IL-7Rα up-regulation by dexamethasone. To further assess the role of Gfi1 in vivo, we generated bacterial artificial chromosome (BAC) transgenic mice, in which a modified Il7r locus expresses GFP to report Il7r gene transcription. By introducing this BAC reporter transgene into either Gfi1-deficient or Gfi1-transgenic mice, we document in vivo that IL-7Rα transcription is up-regulated in the absence of Gfi1 and down-regulated when Gfi1 is overexpressed. Strikingly, the in vivo regulatory role of Gfi1 was specific for CD8(+), and not CD4(+) T cells or immature thymocytes. These results identify Gfi1 as a specific transcriptional repressor of the Il7r gene in CD8 T lymphocytes in vivo.

  18. Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor.

    PubMed

    Wang, Haowei; Dodd, Ian B; Dunlap, David D; Shearwin, Keith E; Finzi, Laura

    2013-06-01

    The lytic-lysogenic decision in bacteriophage 186 is governed by the 186 CI repressor protein in a unique way. The 186 CI is proposed to form a wheel-like oligomer that can mediate either wrapped or looped nucleoprotein complexes to provide the cooperative and competitive interactions needed for regulation. Although consistent with structural, biochemical and gene expression data, many aspects of this model are based on inference. Here, we use atomic force microscopy (AFM) to reveal the various predicted wrapped and looped species, and new ones, for CI regulation of lytic and lysogenic transcription. Automated AFM analysis showed CI particles of the predicted dimensions on the DNA, with CI multimerization favoured by DNA binding. Measurement of the length of the wrapped DNA segments indicated that CI may move on the DNA, wrapping or releasing DNA on either side of the wheel. Tethered particle motion experiments were consistent with wrapping and looping of DNA by CI in solution, where in contrast to λ repressor, the looped species were exceptionally stable. The CI regulatory system provides an intriguing comparison with that of nucleosomes, which share the ability to wrap and release similar sized segments of DNA. PMID:23620280

  19. A novel strategy to analyze L-tryptophan through allosteric Trp repressor based on rolling circle amplification.

    PubMed

    Zhao, Guojie; Hu, Tianyu; Li, Jun; Wei, Hua; Shang, Hong; Guan, Yifu

    2015-09-15

    Rolling circle amplification (RCA) has been considered as a powerful tool for nucleic acids detection. Here, a novel repressor-RCA-based method for L-tryptophan (L-Trp) detection was developed. This method utilizes the specific interaction between the RCA circular template and the Trp repressor protein (TrpR) involved in trp operon of Escherichia coli (E. coli). In the absence of L-Trp, the TrpR protein could not bind to the RCA template, and the RCA process can be continued. When L-Trp is present, the activated TrpR will bind to the operon sequence on the RCA template and inhibit the RCA reaction. Thus, the concentration of L-Trp is correlated directly with the fluorescent RCA signals. We succeeded in detecting L-Trp in a single step in simple homogeneous reaction system. The detection limit was estimated to be 0.77 μM (S/N=3) with good linearity. The method can unambiguously distinguish L-Trp from other 19 standard amino acids and L-Trp analogs. This strategy is also promising for detecting many small molecules such as other amino acids and carbohydrates.

  20. O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis.

    PubMed

    Zentella, Rodolfo; Hu, Jianhong; Hsieh, Wen-Ping; Matsumoto, Peter A; Dawdy, Andrew; Barnhill, Benjamin; Oldenhof, Harriëtte; Hartweck, Lynn M; Maitra, Sushmit; Thomas, Stephen G; Cockrell, Shelley; Boyce, Michael; Shabanowitz, Jeffrey; Hunt, Donald F; Olszewski, Neil E; Sun, Tai-Ping

    2016-01-15

    The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein-protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors-PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)-that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development.

  1. The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.

    PubMed

    Luebke, Justin L; Shen, Jiangchuan; Bruce, Kevin E; Kehl-Fie, Thomas E; Peng, Hui; Skaar, Eric P; Giedroc, David P

    2014-12-01

    How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR [Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor] represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high-resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60' interprotomer cross-links, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR.

  2. Oligogalacturonide-auxin antagonism does not require posttranscriptional gene silencing or stabilization of auxin response repressors in Arabidopsis.

    PubMed

    Savatin, Daniel V; Ferrari, Simone; Sicilia, Francesca; De Lorenzo, Giulia

    2011-11-01

    α-1-4-Linked oligogalacturonides (OGs) derived from plant cell walls are a class of damage-associated molecular patterns and well-known elicitors of the plant immune response. Early transcript changes induced by OGs largely overlap those induced by flg22, a peptide derived from bacterial flagellin, a well-characterized microbe-associated molecular pattern, although responses diverge over time. OGs also regulate growth and development of plant cells and organs, due to an auxin-antagonistic activity. The molecular basis of this antagonism is still unknown. Here we show that, in Arabidopsis (Arabidopsis thaliana), OGs inhibit adventitious root formation induced by auxin in leaf explants as well as the expression of several auxin-responsive genes. Genetic, biochemical, and pharmacological experiments indicate that inhibition of auxin responses by OGs does not require ethylene, jasmonic acid, and salicylic acid signaling and is independent of RESPIRATORY BURST OXIDASE HOMOLOGUE D-mediated reactive oxygen species production. Free indole-3-acetic acid levels are not noticeably altered by OGs. Notably, OG- as well as flg22-auxin antagonism does not involve any of the following mechanisms: (1) stabilization of auxin-response repressors; (2) decreased levels of auxin receptor transcripts through the action of microRNAs. Our results suggest that OGs and flg22 antagonize auxin responses independently of Aux/Indole-3-Acetic Acid repressor stabilization and of posttranscriptional gene silencing.

  3. Structure and Function of the Su(H)-Hairless Repressor Complex, the Major Antagonist of Notch Signaling in Drosophila melanogaster

    PubMed Central

    Torella, Rubben; Preiss, Anette; Maier, Dieter; Kovall, Rhett A.

    2016-01-01

    Notch is a conserved signaling pathway that specifies cell fates in metazoans. Receptor-ligand interactions induce changes in gene expression, which is regulated by the transcription factor CBF1/Su(H)/Lag-1 (CSL). CSL interacts with coregulators to repress and activate transcription from Notch target genes. While the molecular details of the activator complex are relatively well understood, the structure-function of CSL-mediated repressor complexes is poorly defined. In Drosophila, the antagonist Hairless directly binds Su(H) (the fly CSL ortholog) to repress transcription from Notch targets. Here, we determine the X-ray structure of the Su(H)-Hairless complex bound to DNA. Hairless binding produces a large conformational change in Su(H) by interacting with residues in the hydrophobic core of Su(H), illustrating the structural plasticity of CSL molecules to interact with different binding partners. Based on the structure, we designed mutants in Hairless and Su(H) that affect binding, but do not affect formation of the activator complex. These mutants were validated in vitro by isothermal titration calorimetry and yeast two- and three-hybrid assays. Moreover, these mutants allowed us to solely characterize the repressor function of Su(H) in vivo. PMID:27404588

  4. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots

    PubMed Central

    Rizvi, Noreen F.; Weaver, Jessica D.; Cram, Erin J.; Lee-Parsons, Carolyn W. T.

    2016-01-01

    The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis. PMID:27467510

  5. A novel strategy to analyze L-tryptophan through allosteric Trp repressor based on rolling circle amplification.

    PubMed

    Zhao, Guojie; Hu, Tianyu; Li, Jun; Wei, Hua; Shang, Hong; Guan, Yifu

    2015-09-15

    Rolling circle amplification (RCA) has been considered as a powerful tool for nucleic acids detection. Here, a novel repressor-RCA-based method for L-tryptophan (L-Trp) detection was developed. This method utilizes the specific interaction between the RCA circular template and the Trp repressor protein (TrpR) involved in trp operon of Escherichia coli (E. coli). In the absence of L-Trp, the TrpR protein could not bind to the RCA template, and the RCA process can be continued. When L-Trp is present, the activated TrpR will bind to the operon sequence on the RCA template and inhibit the RCA reaction. Thus, the concentration of L-Trp is correlated directly with the fluorescent RCA signals. We succeeded in detecting L-Trp in a single step in simple homogeneous reaction system. The detection limit was estimated to be 0.77 μM (S/N=3) with good linearity. The method can unambiguously distinguish L-Trp from other 19 standard amino acids and L-Trp analogs. This strategy is also promising for detecting many small molecules such as other amino acids and carbohydrates. PMID:25889351

  6. The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus.

    PubMed

    Luebke, Justin L; Shen, Jiangchuan; Bruce, Kevin E; Kehl-Fie, Thomas E; Peng, Hui; Skaar, Eric P; Giedroc, David P

    2014-12-01

    How cells regulate the bioavailability of utilizable sulfur while mitigating the effects of hydrogen sulfide toxicity is poorly understood. CstR [Copper-sensing operon repressor (CsoR)-like sulfurtransferase repressor] represses the expression of the cst operon encoding a putative sulfide oxidation system in Staphylococcus aureus. Here, we show that the cst operon is strongly and transiently induced by cellular sulfide stress in an acute phase and specific response and that cst-encoded genes are necessary to mitigate the effects of sulfide toxicity. Growth defects are most pronounced when S. aureus is cultured in chemically defined media with thiosulfate (TS) as a sole sulfur source, but are also apparent when cystine is used or in rich media. Under TS growth conditions, cells fail to grow as a result of either unregulated expression of the cst operon in a ΔcstR strain or transformation with a non-inducible C31A/C60A CstR that blocks cst induction. This suggests that the cst operon contributes to cellular sulfide homeostasis. Tandem high-resolution mass spectrometry reveals derivatization of CstR by both inorganic tetrasulfide and an organic persulfide, glutathione persulfide, to yield a mixture of Cys31-Cys60' interprotomer cross-links, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR. PMID:25318663

  7. The Banana Transcriptional Repressor MaDEAR1 Negatively Regulates Cell Wall-Modifying Genes Involved in Fruit Ripening.

    PubMed

    Fan, Zhong-Qi; Kuang, Jian-Fei; Fu, Chang-Chun; Shan, Wei; Han, Yan-Chao; Xiao, Yun-Yi; Ye, Yu-Jie; Lu, Wang-Jin; Lakshmanan, Prakash; Duan, Xue-Wu; Chen, Jian-Ye

    2016-01-01

    Ethylene plays an essential role in many biological processes including fruit ripening via modulation of ethylene signaling pathway. Ethylene Response Factors (ERFs) are key transcription factors (TFs) involved in ethylene perception and are divided into AP2, RAV, ERF, and DREB sub-families. Although a number of studies have implicated the involvement of DREB sub-family genes in stress responses, little is known about their roles in fruit ripening. In this study, we identified a DREB TF with a EAR motif, designated as MaDEAR1, which is a nucleus-localized transcriptional repressor. Expression analysis indicated that MaDEAR1 expression was repressed by ethylene, with reduced levels of histone H3 and H4 acetylation at its regulatory regions during fruit ripening. In addition, MaDEAR1 promoter activity was also suppressed in response to ethylene treatment. More importantly, MaDEAR1 directly binds to the DRE/CRT motifs in promoters of several cell wall-modifying genes including MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 associated with fruit softening during ripening and represses their activities. These data suggest that MaDEAR1 acts as a transcriptional repressor of cell wall-modifying genes, and may be negatively involved in ethylene-mediated ripening of banana fruit. Our findings provide new insights into the involvement of DREB TFs in the regulation of fruit ripening. PMID:27462342

  8. Keeping things quiet: Roles of NuRD and Sin3 co-repressor complexes during mammalian development

    PubMed Central

    McDonel, Patrick; Costello, Ita; Hendrich, Brian

    2010-01-01

    Gene inactivation studies of mammalian histone and DNA-modifying proteins have demonstrated a role for many such proteins in embryonic development. Post-implantation embryonic lethality implies a role for epigenetic factors in differentiation and in development of specific lineages or tissues. However a handful of chromatin-modifying enzymes have been found to be required in pre- or peri-implantation embryos. This is significant as implantation is the time when inner cell mass cells of the blastocyst exit pluripotency and begin to commit to form the various lineages that will eventually form the adult animal. These observations indicate a critical role for chromatin-modifying proteins in the earliest lineage decisions of mammalian development, and/or in the formation of the first embryonic cell types. Recent work has shown that the two major class I histone deacetylase-containing co-repressor complexes, the NuRD and Sin3 complexes, are both required at peri-implantation stages of mouse development, demonstrating the importance of histone deacetylation in cell fate decisions. Over the past 10 years both genetic and biochemical studies have revealed surprisingly divergent roles for these two co-repressors in mammalian cells. In this review we will summarise the evidence that the two major class I histone deacetylase complexes in mammalian cells, the NuRD and Sin3 complexes, play important roles in distinct aspects of embryonic development. PMID:18775506

  9. The Banana Transcriptional Repressor MaDEAR1 Negatively Regulates Cell Wall-Modifying Genes Involved in Fruit Ripening

    PubMed Central

    Fan, Zhong-qi; Kuang, Jian-fei; Fu, Chang-chun; Shan, Wei; Han, Yan-chao; Xiao, Yun-yi; Ye, Yu-jie; Lu, Wang-jin; Lakshmanan, Prakash; Duan, Xue-wu; Chen, Jian-ye

    2016-01-01

    Ethylene plays an essential role in many biological processes including fruit ripening via modulation of ethylene signaling pathway. Ethylene Response Factors (ERFs) are key transcription factors (TFs) involved in ethylene perception and are divided into AP2, RAV, ERF, and DREB sub-families. Although a number of studies have implicated the involvement of DREB sub-family genes in stress responses, little is known about their roles in fruit ripening. In this study, we identified a DREB TF with a EAR motif, designated as MaDEAR1, which is a nucleus-localized transcriptional repressor. Expression analysis indicated that MaDEAR1 expression was repressed by ethylene, with reduced levels of histone H3 and H4 acetylation at its regulatory regions during fruit ripening. In addition, MaDEAR1 promoter activity was also suppressed in response to ethylene treatment. More importantly, MaDEAR1 directly binds to the DRE/CRT motifs in promoters of several cell wall-modifying genes including MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 associated with fruit softening during ripening and represses their activities. These data suggest that MaDEAR1 acts as a transcriptional repressor of cell wall-modifying genes, and may be negatively involved in ethylene-mediated ripening of banana fruit. Our findings provide new insights into the involvement of DREB TFs in the regulation of fruit ripening. PMID:27462342

  10. FHL2 interacts with and acts as a functional repressor of Id2 in human neuroblastoma cells

    PubMed Central

    Han, Weidong; Wu, Zhiqiang; Zhao, Yali; Meng, Yuanguang; Si, Yiling; Yang, Jie; Fu, Xiaobing; Yu, Li

    2009-01-01

    Inhibitor of differentiation 2 (Id2) is a natural inhibitor of the basic helix–loop–helix transcription factors. Although Id2 is well known to prevent differentiation and promote cell-cycle progression and tumorigenesis, the molecular events that regulate Id2 activity remain to be investigated. Here, we identified that Four-and-a-half LIM-only protein 2 (FHL2) is a novel functional repressor of Id2. Moreover, we demonstrated that FHL2 can directly interact with all members of the Id family (Id1–4) via an N-terminal loop–helix structure conserved in Id proteins. FHL2 antagonizes the inhibitory effect of Id proteins on basic helix–loop–helix protein E47-mediated transcription, which was abrogated by the deletion mutation of Ids that disrupted their interaction with FHL2. We also showed a competitive nature between FHL2 and E47 for binding Id2, whereby FHL2 prevents the formation of the Id2–E47 heterodimer, thus releasing E47 to DNA and restoring its transcriptional activity. FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells, during which the expression of Id2 was opposite to that. Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2. Altogether, these results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells. PMID:19417068

  11. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

    PubMed

    Rizvi, Noreen F; Weaver, Jessica D; Cram, Erin J; Lee-Parsons, Carolyn W T

    2016-01-01

    The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis. PMID:27467510

  12. A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-β mediated epithelial-mesenchymal transition

    PubMed Central

    Vincent, Theresa; Neve, Etienne P. A.; Johnson, Jill R.; Kukalev, Alexander; Rojo, Federico; Albanell, Joan; Pietras, Kristian; Virtanen, Ismo; Philipson, Lennart; Leopold, Philip L.; Crystal, Ronald G.; de Herreros, Antonio Garcia; Moustakas, Aristidis; Pettersson, Ralf F.; Fuxe, Jonas

    2013-01-01

    Epithelial-mesenchymal transitions (EMT) are essential for organogenesis and triggered in carcinoma progression into an invasive state1. Transforming growth factor-β (TGF-β) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT2-5, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT6, 7. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight junction protein, and E-cadherin during TGF-β-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited the repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a novel mechanism of gene repression during EMT. PMID:19597490

  13. Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of purB, purC, purL, purMN and guaBA expression in Escherichia coli.

    PubMed

    Meng, L M; Kilstrup, M; Nygaard, P

    1990-01-26

    The purR gene encodes a repressor (PurR) controlling the synthesis of the enzymes of purine biosynthesis. The subunit of PurR was identified as a 38-kDa polypeptide by SDS/polyacrylamide gel electrophoresis. Analysis of a purR-lacZ transcriptional fusion indicated that purR expression is autoregulated. This was confirmed by gel retardation and DNaseI footprinting experiments, where two PurR-binding sites were identified in the transcribed part of purR. Introduction of a purR mutation in wild-type and pur-lac fusion strains was found to abolish purine repression of all genes of the purine biosynthetic pathway except for purA.

  14. The Arabidopsis repressor of light signaling SPA1 acts in the phloem to regulate seedling de-etiolation, leaf expansion and flowering time.

    PubMed

    Ranjan, Aashish; Fiene, Gabriele; Fackendahl, Petra; Hoecker, Ute

    2011-05-01

    Plants adjust their growth and development in response to the ambient light environment. These light responses involve systemic signals that coordinate differentiation of different tissues and organs. Here, we have investigated the function of the key repressor of photomorphogenesis SPA1 in different tissues of the plant by expressing GUS-SPA1 under the control of tissue-specific promoters in a spa mutant background. We show that SPA1 expression in the phloem vasculature is sufficient to rescue the spa1 mutant phenotype in dark-grown spa mutant seedlings. Expression of SPA1 in mesophyll, epidermis or root tissues of the seedling, by contrast, has no or only slight effects. In the leaf, SPA1 expression in both the phloem and the mesophyll is required for full complementation of the defect in leaf expansion. SPA1 in phloem and mesophyll tissues affected division and expansion of cells in the epidermal layer, indicating that SPA1 induces non-cell-autonomous responses also in the leaf. Photoperiodic flowering is exclusively controlled by SPA1 expression in the phloem, which is consistent with previous results showing that the direct substrate of the COP1/SPA complex, CONSTANS, also acts in the phloem. Taken together, our results highlight the importance of phloem vascular tissue in coordinating growth and development. Because the SPA1 protein itself is incapable of moving from cell to cell, we suggest that SPA1 regulates the activity of downstream component(s) of light signaling that subsequently act in a non-cell-autonomous manner. SPA1 action in the phloem may also result in mechanical stimuli that affect cell elongation and cell division in other tissues.

  15. Differential regulation of high-affinity phosphate transport systems of Mycobacterium smegmatis: identification of PhnF, a repressor of the phnDCE operon.

    PubMed

    Gebhard, Susanne; Cook, Gregory M

    2008-02-01

    The uptake of phosphate into the cell via high-affinity, phosphate-specific transport systems has been studied with several species of mycobacteria. All of these species have been shown to contain several copies of such transport systems, which are synthesized in response to phosphate limitation. However, the mechanisms leading to the expression of the genes encoding these transporters have not been studied. This study reports on the investigation of the regulation of the pstSCAB and the phnDCE operons of Mycobacterium smegmatis. The phn locus contains an additional gene, phnF, encoding a GntR-like transcriptional regulator. Expression analyses of a phnF deletion mutant demonstrated that PhnF acts as a repressor of the phnDCE operon but does not affect the expression of pstSCAB. The deletion of pstS, which is thought to cause the constitutive expression of genes regulated by the two-component system SenX3-RegX3, led to the constitutive expression of the transcriptional fusions pstS-lacZ, phnD-lacZ, and phnF-lacZ, suggesting that phnDCE and phnF are conceivably new members of the SenX3-RegX3 regulon of M. smegmatis. Two presumptive binding sites for PhnF in the intergenic region between phnD and phnF were identified and shown to be required for the repression of phnD and phnF, respectively. We propose a model in which the transcription of pstSCAB is controlled by the two-component SenX3-RegX3 system, while phnDCE and phnF are subject to dual control by SenX3-RegX3 and PhnF. PMID:18083811

  16. Structure-based functional characterization of repressor of toxin (Rot), a central regulator of staphylococcus aureus virulence

    SciTech Connect

    Killikelly, April; Jakoncic, Jean; Benson, Meredith A.; Ohneck, Elizabeth A.; Sampson, Jared M.; Spurrier, Brett; Torres, Victoer J.; Kong, Xian -Peng

    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 of 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.

  17. The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

    PubMed Central

    Nikel, Pablo I.; Romero-Campero, Francisco J.; Zeidman, Joshua A.; Goñi-Moreno, Ángel

    2015-01-01

    ABSTRACT The growth of the soil bacterium Pseudomonas putida KT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(glpD-gfp) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of the glp genes. Either deleting glpR (encoding the G3P-responsive transcriptional repressor that controls the expression of the glpFKRD gene cluster) or altering G3P formation (by overexpressing glpK, encoding glycerol kinase) abolished the bimodal glpD expression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision of glpR in trans restored the phenotypes lost in the ΔglpR mutant. The prolonged nongrowth regime of P. putida on glycerol could thus be traced to the regulatory device controlling the transcription of the glp genes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states. PMID:25827416

  18. Ion concentration and temperature dependence of DNA binding: comparison of PurR and LacI repressor proteins.

    PubMed

    Moraitis, M I; Xu, H; Matthews, K S

    2001-07-10

    Purine repressor (PurR) binding to specific DNA is enhanced by complexing with purines, whereas lactose repressor (LacI) binding is diminished by interaction with inducer sugars despite 30% identity in their protein sequences and highly homologous tertiary structures. Nonetheless, in switching from low- to high-affinity DNA binding, these proteins undergo a similar structural change in which the hinge region connecting the DNA and effector binding domains folds into an alpha-helix and contacts the DNA minor groove. The differences in response to effector for these proteins should be manifest in the polyelectrolyte effect which arises from cations displaced from DNA by interaction with positively charged side chains on a protein and is quantitated by measurement of DNA binding affinity as a function of ion concentration. Consistent with structural data for these proteins, high-affinity operator DNA binding by the PurR-purine complex involved approximately 15 ion pairs, a value significantly greater than that for the corresponding state of LacI (approximately 6 ion pairs). For both proteins, however, conversion to the low-affinity state results in a decrease of approximately 2-fold in the number of cations released per dimeric DNA binding site. Heat capacity changes (DeltaC(p)) that accompany DNA binding, derived from buried apolar surface area, coupled folding, and restriction of motional freedom of polar groups in the interface, also reflect the differences between these homologous repressor proteins. DNA binding of the PurR-guanine complex is accompanied by a DeltaC(p) (-2.8 kcal mol(-1) K(-1)) more negative than that observed previously for LacI (-0.9 to -1.5 kcal mol(-1) K(-1)), suggesting that more extensive protein folding and/or enhanced structural rigidity may occur upon DNA binding for PurR compared to DNA binding for LacI. The differences between these proteins illustrate plasticity of function despite high-level sequence and structural homology and

  19. Corynebacterium glutamicum Zur acts as a zinc-sensing transcriptional repressor of both zinc-inducible and zinc-repressible genes involved in zinc homeostasis.

    PubMed

    Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2012-12-01

    Zur is a zinc-dependent transcriptional repressor of zinc uptake systems in bacteria. In the present study, we examined the role of Corynebacterium glutamicum Zur in the zinc-inducible expression of two genes: one encoding a cation diffusion facilitator (zrf) and the other a metal-translocating P-type ATPase (zra). Both genes were shown to be involved in zinc resistance. Disruption of the zur gene encoding Zur resulted in constitutive expression of zrf and zra mRNAs. An electrophoretic mobility shift assay revealed that the Zur protein binds to the zrf and zra promoters, for which the in vivo activities were up-regulated in response to excess zinc. Interestingly, the in vitro DNA binding activity of Zur was inhibited by zinc, in contrast to its zinc-dependent binding to the promoter region of a zinc-repressible ABC transporter gene znuB2. A 21-bp motif found in the Zur binding site overlaps the putative -35 region of both the zrf and zra promoters. This new motif is a 10-1-10 direct repeat sequence distinct from the 10-1-10 inverted repeat sequence of a previously identified Zur box for zinc-dependent binding. Nevertheless, their 10-bp elements share some sequence similarities. Overexpression of zur in the zur deletion mutant background, as well as deletion of zur in the zrf and zra double deletion mutant background, resulted in decreased resistance to zinc. These results suggest that the direct negative control of both zinc uptake and export systems by Zur is central to C. glutamicum zinc homeostasis and is effected in distinct ways. PMID:23061624

  20. Kaiso, a transcriptional repressor, promotes cell migration and invasion of prostate cancer cells through regulation of miR-31 expression.

    PubMed

    Wang, Honghe; Liu, Wei; Black, ShaNekkia; Turner, Omari; Daniel, Juliet M; Dean-Colomb, Windy; He, Qinghua P; Davis, Melissa; Yates, Clayton

    2016-02-01

    Kaiso, a member of the BTB/POZ zinc finger protein family, functions as a transcriptional repressor by binding to sequence-specific Kaiso binding sites or to methyl-CpG dinucleotides. Previously, we demonstrated that Kaiso overexpression and nuclear localization correlated with the progression of prostate cancer (PCa). Therefore, our objective was to explore the molecular mechanisms underlying Kaiso-mediated PCa progression. Comparative analysis of miRNA arrays revealed that 13 miRNAs were significantly altered (> 1.5 fold, p < 0.05) in sh-Kaiso PC-3 compared to sh-Scr control cells. Real-time PCR validated that three miRNAs (9, 31, 636) were increased in sh-Kaiso cells similar to cells treated with 5-aza-2'-deoxycytidine. miR-31 expression negatively correlated with Kaiso expression and with methylation of the miR-31 promoter in a panel of PCa cell lines. ChIP assays revealed that Kaiso binds directly to the miR-31 promoter in a methylation-dependent manner. Over-expression of miR-31 decreased cell proliferation, migration and invasiveness of PC-3 cells, whereas cells transfected with anti-miR-31 restored proliferation, migration and invasiveness of sh-Kaiso PC-3 cells. In PCa patients, Kaiso high/miR-31 low expression correlated with worse overall survival relative to each marker individually. In conclusion, these results demonstrate that Kaiso promotes cell migration and invasiveness through regulation of miR-31 expression.

  1. Proteasomes, Sir2, and Hxk2 form an interconnected aging network that impinges on the AMPK/Snf1-regulated transcriptional repressor Mig1.

    PubMed

    Yao, Yanhua; Tsuchiyama, Scott; Yang, Ciyu; Bulteau, Anne Laure; He, Chong; Robison, Brett; Tsuchiya, Mitsuhiro; Miller, Delana; Briones, Valeria; Tar, Krisztina; Potrero, Anahi; Friguet, Bertrand; Kennedy, Brian K; Schmidt, Marion

    2015-01-01

    Elevated proteasome activity extends lifespan in model organisms such as yeast, worms and flies. This pro-longevity effect might be mediated by improved protein homeostasis, as this protease is an integral module of the protein homeostasis network. Proteasomes also regulate cellular processes through temporal and spatial degradation of signaling pathway components. Here we demonstrate that the regulatory function of the proteasome plays an essential role in aging cells and that the beneficial impact of elevated proteasome capacity on lifespan partially originates from deregulation of the AMPK signaling pathway. Proteasome-mediated lifespan extension activity was carbon-source dependent and cells with enhancement proteasome function exhibited increased respiratory activity and oxidative stress response. These findings suggested that the pro-aging impact of proteasome upregulation might be related to changes in the metabolic state through a premature induction of respiration. Deletion of yeast AMPK, SNF1, or its activator SNF4 abrogated proteasome-mediated lifespan extension, supporting this hypothesis as the AMPK pathway regulates metabolism. We found that the premature induction of respiration in cells with increased proteasome activity originates from enhanced turnover of Mig1, an AMPK/Snf1 regulated transcriptional repressor that prevents the induction of genes required for respiration. Increasing proteasome activity also resulted in partial relocation of Mig1 from the nucleus to the mitochondria. Collectively, the results argue for a model in which elevated proteasome activity leads to the uncoupling of Snf1-mediated Mig1 regulation, resulting in a premature activation of respiration and thus the induction of a mitohormetic response, beneficial to lifespan. In addition, we observed incorrect Mig1 localization in two other long-lived yeast aging models: cells that overexpress SIR2 or deleted for the Mig1-regulator HXK2. Finally, compromised proteasome function

  2. Vaccinia virus morphogenesis is interrupted when expression of the gene encoding an 11-kilodalton phosphorylated protein is prevented by the Escherichia coli lac repressor.

    PubMed

    Zhang, Y F; Moss, B

    1991-11-01

    A conditional lethal vaccinia virus mutant, which constitutively expresses the Escherichia coli lac repressor and has the lac operator controlling the F18R gene (the 18th open reading frame of the HindIII F fragment of the vaccinia virus strain WR genome) encoding an 11-kDa protein, was previously shown to be dependent on the inducer isopropyl-beta-D-thiogalactoside (IPTG) for replication (Y. Zhang and B. Moss, Proc. Natl. Acad. Sci. USA 88:1511-1515, 1991). Further studies indicated that the yield of infectious virus could be regulated by titration with IPTG and that virus production was arrested by IPTG removal at appropriate times. Under nonpermissive conditions, an 11-kDa protein reactive with antiserum raised to a previously described DNA-binding phosphoprotein (S. Y. Kao and W. R. Bauer, Virology 159:399-407, 1987) was not synthesized, indicating that the latter is the product of the F18R gene. In the absence of IPTG, replication of viral DNA and the subsequent resolution of concatemeric DNA molecules appeared normal. Omission of IPTG did not alter the kinetics of early and late viral protein synthesis, although the absence of the 11-kDa polypeptide was noted by labeling infected cells with [35S]methionine or [32P]phosphate. Pulse-chase experiments revealed that proteolytic processing of the major viral structural proteins, P4a and P4b, was inhibited under nonpermissive conditions, suggesting a block in virus maturation. Without addition of IPTG, the failure of virus particle formation was indicated by sucrose gradient centrifugation of infected cell lysates and by the absence of vaccinia virus-mediated pH-dependent cell fusion. Electron microscopic examination of infected cells revealed that immature virus particles, with aberrant internal structures, accumulated when synthesis of the 11-kDa DNA-binding protein was prevented.

  3. The translational repressor eIF4E-binding protein 2 (4E-BP2) correlates with selective delayed neuronal death after ischemia

    PubMed Central

    Ayuso, María Irene; Martínez-Alonso, Emma; Cid, Cristina; de Leciñana, Maria Alonso; Alcázar, Alberto

    2013-01-01

    Transient brain ischemia induces an inhibition of translational rates and causes delayed neuronal death in selective regions and cognitive deficits, whereas these effects do not occur in resistant areas. The translational repressor eukaryotic initiation factor (eIF) 4E-binding protein-2 (4E-BP2) specifically binds to eIF4E and is critical in the control of protein synthesis. To link neuronal death to translation inhibition, we study the eIF4E association with 4E-BP2 under ischemia reperfusion in a rat model of transient forebrain ischemia. Upon reperfusion, a selective neuronal apoptosis in the hippocampal cornu ammonis 1 (CA1) region was induced, while it did not occur in the cerebral cortex. Confocal microscopy analysis showed a decrease in 4E-BP2/eIF4E colocalization in resistant cortical neurons after reperfusion. In contrast, in vulnerable CA1 neurons, 4E-BP2 remains associated to eIF4E with a higher degree of 4E-BP2/eIF4E colocalization and translation inhibition. Furthermore, the binding of a 4E-BP2 peptide to eIF4E induced neuronal apoptosis in the CA1 region. Finally, pharmacological-induced protection of CA1 neurons inhibited neuronal apoptosis, decreased 4E-BP2/eIF4E association, and recovered translation. These findings documented specific changes in 4E-BP2/eIF4E association during ischemic reperfusion, linking the translation inhibition to selective neuronal death, and identifying 4E-BP2 as a novel target for protection of vulnerable neurons in ischemic injury. PMID:23591646

  4. LitR Is a Repressor of syp Genes and Has a Temperature-Sensitive Regulatory Effect on Biofilm Formation and Colony Morphology in Vibrio (Aliivibrio) salmonicida

    PubMed Central

    Bjelland, Ane Mohn; Ronessen, Maria; Robertsen, Espen; Willassen, Nils Peder

    2014-01-01

    Vibrio (Aliivibrio) salmonicida is the etiological agent of cold water vibriosis, a disease in farmed Atlantic salmon (Salmo salar) that is kept under control due to an effective vaccine. A seawater temperature below 12°C is normally required for disease development. Quorum sensing (QS) is a cell density-regulated communication system that bacteria use to coordinate activities involved in colonization and pathogenesis, and we have previously shown that inactivation of the QS master regulator LitR attenuates the V. salmonicida strain LFI1238 in a fish model. We show here that strain LFI1238 and a panel of naturally occurring V. salmonicida strains are poor biofilm producers. Inactivation of litR in the LFI1238 strain enhances medium- and temperature-dependent adhesion, rugose colony morphology, and biofilm formation. Chemical treatment and electron microscopy of the biofilm identified an extracellular matrix consisting mainly of a fibrous network, proteins, and polysaccharides. Further, by microarray analysis of planktonic and biofilm cells, we identified a number of genes regulated by LitR and, among these, were homologues of the Vibrio fischeri symbiosis polysaccharide (syp) genes. The syp genes were regulated by LitR in both planktonic and biofilm lifestyle analyses. Disruption of syp genes in the V. salmonicida ΔlitR mutant alleviated adhesion, rugose colony morphology, and biofilm formation. Hence, LitR is a repressor of syp transcription that is necessary for expression of the phenotypes examined. The regulatory effect of LitR on colony morphology and biofilm formation is temperature sensitive and weak or absent at temperatures above the bacterium's upper threshold for pathogenicity. PMID:24973072

  5. The Transcriptional Repressor ID2 Can Interact with the Canonical Clock Components CLOCK and BMAL1 and Mediate Inhibitory Effects on mPer1 Expression*

    PubMed Central

    Ward, Sarah M.; Fernando, Shanik J.; Hou, Tim Y.; Duffield, Giles E.

    2010-01-01

    ID2 is a rhythmically expressed HLH transcriptional repressor. Deletion of Id2 in mice results in circadian phenotypes, highlighted by disrupted locomotor activity rhythms and an enhanced photoentrainment response. ID2 can suppress the transactivation potential of the positive elements of the clock, CLOCK-BMAL1, on mPer1 and clock-controlled gene (CCG) activity. Misregulation of CCGs is observed in Id2−/− liver, and mutant mice exhibit associated alterations in lipid homeostasis. These data suggest that ID2 contributes to both input and output components of the clock and that this may be via interaction with the bHLH clock proteins CLOCK and BMAL1. The aim of the present study was to explore this potential interaction. Coimmunoprecipitation analysis revealed the capability of ID2 to complex with both CLOCK and BMAL1, and mammalian two-hybrid analysis revealed direct interactions of ID2, ID1 and ID3 with CLOCK and BMAL1. Deletion of the ID2 HLH domain rendered ID2 ineffective at inhibiting CLOCK-BMAL1 transactivation, suggesting that interaction between the proteins is via the HLH region. Immunofluorescence analysis revealed overlapping localization of ID2 with CLOCK and BMAL1 in the cytoplasm. Overexpression of CLOCK and BMAL1 in the presence of ID2 resulted in a significant reduction in their nuclear localization, revealing that ID2 can sequester CLOCK and BMAL1 to the cytoplasm. Serum stimulation of Id2−/− mouse embryonic fibroblasts resulted in an enhanced induction of mPer1 expression. These data provide the basis for a molecular mechanism through which ID2 could regulate aspects of both clock input and output through a time-of-day specific interaction with CLOCK and BMAL1. PMID:20861012

  6. The Arabidopsis SWI2/SNF2 chromatin Remodeler BRAHMA regulates polycomb function during vegetative development and directly activates the flowering repressor gene SVP.

    PubMed

    Li, Chenlong; Chen, Chen; Gao, Lei; Yang, Songguang; Nguyen, Vi; Shi, Xuejiang; Siminovitch, Katherine; Kohalmi, Susanne E; Huang, Shangzhi; Wu, Keqiang; Chen, Xuemei; Cui, Yuhai

    2015-01-01

    The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.

  7. Kaiso, a transcriptional repressor, promotes cell migration and invasion of prostate cancer cells through regulation of miR-31 expression

    PubMed Central

    Wang, Honghe; Liu, Wei; Black, ShaNekkia; Turner, Omari; Daniel, Juliet M.; Dean-Colomb, Windy; He, Qinghua P.; Davis, Melissa; Yates, Clayton

    2016-01-01

    Kaiso, a member of the BTB/POZ zinc finger protein family, functions as a transcriptional repressor by binding to sequence-specific Kaiso binding sites or to methyl-CpG dinucleotides. Previously, we demonstrated that Kaiso overexpression and nuclear localization correlated with the progression of prostate cancer (PCa). Therefore, our objective was to explore the molecular mechanisms underlying Kaiso-mediated PCa progression. Comparative analysis of miRNA arrays revealed that 13 miRNAs were significantly altered (> 1.5 fold, p < 0.05) in sh-Kaiso PC-3 compared to sh-Scr control cells. Real-time PCR validated that three miRNAs (9, 31, 636) were increased in sh-Kaiso cells similar to cells treated with 5-aza-2′-deoxycytidine. miR-31 expression negatively correlated with Kaiso expression and with methylation of the miR-31 promoter in a panel of PCa cell lines. ChIP assays revealed that Kaiso binds directly to the miR-31 promoter in a methylation-dependent manner. Over-expression of miR-31 decreased cell proliferation, migration and invasiveness of PC-3 cells, whereas cells transfected with anti-miR-31 restored proliferation, migration and invasiveness of sh-Kaiso PC-3 cells. In PCa patients, Kaiso high/miR-31 low expression correlated with worse overall survival relative to each marker individually. In conclusion, these results demonstrate that Kaiso promotes cell migration and invasiveness through regulation of miR-31 expression. PMID:26734997

  8. The transcription factor MAZR preferentially acts as a transcriptional repressor in mast cells and plays a minor role in the regulation of effector functions in response to FcεRI stimulation.

    PubMed

    Abramova, Anastasia; Sakaguchi, Shinya; Schebesta, Alexandra; Hassan, Hammad; Boucheron, Nicole; Valent, Peter; Roers, Axel; Ellmeier, Wilfried

    2013-01-01

    Mast cells are key players in type I hypersensitivity reactions in humans and mice and their activity has to be tightly controlled. Previous studies implicated the transcription factor MAZR in the regulation of mast cell function. To study the role of MAZR in mast cells, we generated a conditional Mazr allele and crossed Mazr (F/F) mice with the Vav-iCre deleter strain, which is active in all hematopoietic cells. MAZR-null BM-derived mast cells (BMMC) were phenotypically indistinguishable from wild-type BMMCs, although the numbers of IL-3 generated Mazr (F/F) Vav-iCre BMMCs were reduced in comparison to Mazr (F/F) BMMCs, showing that MAZR is required for the efficient generation of BMMC in vitro. A gene expression analysis revealed that MAZR-deficiency resulted in the dysregulation of 128 genes, with more genes up- than down-regulated in the absence of MAZR, indicating that MAZR acts as a transcriptional repressor in mast cells. Among the up-regulated genes were the chemokines Ccl5, Cxcl10, Cxcl12, the chemokine receptor Ccr5 and the cytokine IL18, suggesting an immunoregulatory role for MAZR in mast cells. Enforced expression of MAZR in mature Mazr-deficient BMMCs rescued the altered expression pattern of some genes tested, suggesting direct regulation of these genes by MAZR. Upon FcεRI stimulation, Mazr expression was transiently down-regulated in BMMCs. However, early and late effector functions in response to FcεRI-mediated stimulation were not impaired in the absence of MAZR, with the exception of IL-6, which was slightly decreased. Taken together, out data indicate that MAZR preferentially acts as a transcriptional repressor in mast cells, however MAZR plays only a minor role in the transcriptional networks that regulate early and late effector functions in mast cells in response to FcεRI stimulation.

  9. An NRSF/REST-like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Drosophila

    PubMed Central

    Tsuda, Leo; Kaido, Masako; Lim, Young-Mi; Kato, Kagayaki; Aigaki, Toshiro; Hayashi, Shigeo

    2006-01-01

    The corepressor complex that includes Ebi and SMRTER is a target of epidermal growth factor (EGF) and Notch signaling pathways and regulates Delta (Dl)-mediated induction of support cells adjacent to photoreceptor neurons of the Drosophila eye. We describe a mechanism by which the Ebi/SMRTER corepressor complex maintains Dl expression. We identified a gene, charlatan (chn), which encodes a C2H2-type zinc-finger protein resembling human neuronal restricted silencing factor/repressor element RE-1 silencing transcription factor (NRSF/REST). The Ebi/SMRTER corepressor complex represses chn transcription by competing with the activation complex that includes the Notch intracellular domain (NICD). Chn represses Dl expression and is critical for the initiation of eye development. Thus, under EGF signaling, double negative regulation mediated by the Ebi/SMRTER corepressor complex and an NRSF/REST-like factor, Chn, maintains inductive activity in developing photoreceptor cells by promoting Dl expression. PMID:16763555

  10. The transcriptional repressor Blimp1 is expressed in rare luminal progenitors and is essential for mammary gland development.

    PubMed

    Ahmed, Mohammed I; Elias, Salah; Mould, Arne W; Bikoff, Elizabeth K; Robertson, Elizabeth J

    2016-05-15

    Mammary gland morphogenesis depends on a tight balance between cell proliferation, differentiation and apoptosis, to create a defined functional hierarchy within the epithelia. The limited availability of stem cell/progenitor markers has made it challenging to decipher lineage relationships. Here, we identify a rare subset of luminal progenitors that express the zinc finger transcriptional repressor Blimp1, and demonstrate that this subset of highly clonogenic luminal progenitors is required for mammary gland development. Conditional inactivation experiments using K14-Cre and WAPi-Cre deleter strains revealed essential functions at multiple developmental stages. Thus, Blimp1 regulates proliferation, apoptosis and alveolar cell maturation during puberty and pregnancy. Loss of Blimp1 disrupts epithelial architecture and lumen formation both in vivo and in three-dimensional (3D) primary cell cultures. Collectively, these results demonstrate that Blimp1 is required to maintain a highly proliferative luminal subset necessary for mammary gland development and homeostasis.

  11. Noncoding RNA Gas5 Is a Growth Arrest and Starvation-Associated Repressor of the Glucocorticoid Receptor

    PubMed Central

    Kino, Tomoshige; Hurt, Darrell E.; Ichijo, Takamasa; Nader, Nancy; Chrousos, George P.

    2010-01-01

    The availability of nutrients influences cellular growth and survival by affecting gene transcription. Glucocorticoids also influence gene transcription and have diverse activities on cell growth, energy expenditure, and survival. We found that the growth arrest-specific 5 (Gas5) noncoding RNA, which is abundant in cells whose growth has been arrested due to lack of nutrients or growth factors, sensitized cells to apoptosis by suppressing glucocorticoid-mediated induction of several responsive genes, including the one encoding cellular inhibitor of apoptosis 2. Gas5 bound to the DNA-binding domain of the glucocorticoid receptor (GR) by acting as a decoy “glucocorticoid response element (GRE)”, thus, competing with DNA GREs for binding to the GR. We conclude that Gas5 is a ribo-repressor of the GR, influencing cell survival and metabolic activities during starvation by modulating the transcriptional activity of the GR. PMID:20124551

  12. Identification of amino acids in lac repressor protein cross-linked to operator DNA specifically substituted with bromodeoxyuridine.

    PubMed

    Allen, T D; Wick, K L; Matthews, K S

    1991-04-01

    Amino acids in lac repressor protein which form cross-links to lac operator DNA specifically substituted with bromodeoxyuridine (BrdU) have been identified. Five sites of cross-linking in BrdU-substituted operator DNA were found at positions +3, +4, +14, +18, and +19 relative to the initiation site for transcription (Wick, K.L., and Matthews, K.S. (1991) J. Biol. Chem. 266, 6106-6112). Irradiation of complexes of repressor and each of these five singly substituted operator DNAs was executed under large scale conditions to generate sufficient complex for proteolysis, separation of the peptide-DNA, and peptide sequencing. The DNAs substituted with BrdU for thymidine at positions +3, +18, and +19 yielded cross-links to the peptide spanning residues 23-33, with the cross-link identified at His-29. Substitution at position +14 resulted in a cross-link to Tyr-17 within the peptide containing amino acids 13-22. These results are consistent with the structure determined by NMR and molecular dynamics calculations of the NH2-terminal headpiece-symmetric operator complex (Lamerichs, R.M.J.N., Boelens, R., van der Marel, G.A., van Boom, J.H., Kaptein, R., Buck, F., Fera, B., and Rüterjans, H. (1989) Biochemistry 28, 2895-2991; de Vlieg, J., Berendsen, H.J.C., and van Gunsteren, W.F. (1989) Proteins 6, 104-127). This structure indicates proximity of His-29 in the major groove to thymidines at positions +3 and +4. Since base pairs at positions +18 and +19 occupy symmetrical positions to +3 and +4 in the promoter distal region of the operator, it would be anticipated that cross-links similar to the +3 and +4 positions would form at these sites; this prediction is not borne out by the behavior at +4/+18, as no peptide could be identified cross-linked to DNA substituted at +4. Molecular dynamics simulations and the NMR data indicate that Tyr-17 interacts with the thymine at position +8, which is symmetrically related to position +14. Although BrdU-associated strand scission at +8

  13. The transcriptional repressor Blimp1 is expressed in rare luminal progenitors and is essential for mammary gland development

    PubMed Central

    Ahmed, Mohammed I.; Mould, Arne W.; Bikoff, Elizabeth K.

    2016-01-01

    ABSTRACT Mammary gland morphogenesis depends on a tight balance between cell proliferation, differentiation and apoptosis, to create a defined functional hierarchy within the epithelia. The limited availability of stem cell/progenitor markers has made it challenging to decipher lineage relationships. Here, we identify a rare subset of luminal progenitors that express the zinc finger transcriptional repressor Blimp1, and demonstrate that this subset of highly clonogenic luminal progenitors is required for mammary gland development. Conditional inactivation experiments using K14-Cre and WAPi-Cre deleter strains revealed essential functions at multiple developmental stages. Thus, Blimp1 regulates proliferation, apoptosis and alveolar cell maturation during puberty and pregnancy. Loss of Blimp1 disrupts epithelial architecture and lumen formation both in vivo and in three-dimensional (3D) primary cell cultures. Collectively, these results demonstrate that Blimp1 is required to maintain a highly proliferative luminal subset necessary for mammary gland development and homeostasis. PMID:27190036

  14. A nuclear magnetic resonance study of the DNA-binding affinity of Cro repressor protein stabilized by a disulfide bond.

    PubMed

    Baleja, J D; Sykes, B D

    1994-01-01

    The structure, dynamics, and DNA-binding characteristics of wild-type and cross-linked Cro repressors are compared by using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The Cro repressor is a small dimeric DNA-binding protein from bacteriophage lambda. Replacement of valine-55 by cysteine in the dimer interaction region of each monomer subunit results in the spontaneous formation of a disulfide cross-link between the subunits. Two-dimensional nuclear Overhauser effect spectroscopy and CD data show the variant has nearly the same conformation as the wild-type protein. However, by monitoring the CD band at 222 nm, the cross-linked protein is shown to have a heat-denaturation midpoint temperature of 67 degrees C, whereas the wild-type protein has a melting temperature of about 47 degrees C. Using 1H-NMR to follow the denaturation by heat, the same melting temperature is observed for wild-type Cro (47 degrees C), but a much lower melting temperature is seen for V55C Cro (58 degrees C). This suggests that between 58 and 67 degrees C the cross-linked protein exists in a molten globule state with the alpha-helices mainly intact, but without the interaction of chemical groups that cause spectral dispersion. Binding parameters for interaction of the proteins with DNA were obtained by observing the NMR spectrum for the imino protons of a 10 base-pair half-operator DNA and titrating in protein. The cross-linked protein binds DNA (Kd = 160 microM) about eight times more weakly than the wild-type protein (Kd = 19 microM). Adjustments in protein structure, necessary to form a tight protein-DNA complex, appear to be hindered by a loss in protein flexibility caused by the intersubunit cross-link.

  15. The product of the murine homolog of the Drosophila extra sex combs gene displays transcriptional repressor activity.

    PubMed Central

    Denisenko, O N; Bomsztyk, K

    1997-01-01

    The heterogeneous nuclear ribonucleoprotein K protein represents a novel class of proteins that may act as docking platforms that orchestrate cross-talk among molecules involved in signal transduction and gene expression. Using a fragment of K protein as bait in the yeast two-hybrid screen, we isolated a cDNA that encodes a protein whose primary structure has extensive similarity to the Drosophila melanogaster extra sex combs (esc) gene product, Esc, a putative silencer of homeotic genes. The cDNA that we isolated is identical to the cDNA of the recently positionally cloned mouse embryonic ectoderm development gene, eed. Like Esc, Eed contains six WD-40 repeats in the C-terminal half of the protein and is thought to repress homeotic gene expression during mouse embryogenesis. Eed binds to K protein through a domain in its N terminus, but interestingly, this domain is not found in the Drosophila Esc. Gal4-Eed fusion protein represses transcription of a reporter gene driven by a promoter that contains Gal4-binding DNA elements. Eed also represses transcription when recruited to a target promoter by Gal4-K protein. Point mutations within the eed gene that are responsible for severe embryonic development abnormalities abolished the transcriptional repressor activity of Eed. Results of this study suggest that Eed-restricted homeotic gene expression during embryogenesis reflects the action of Eed as a transcriptional repressor. The Eed-mediated transcriptional effects are likely to reflect the interaction of Eed with multiple molecular partners, including K protein. PMID:9234727

  16. Tcf7l2/Tcf4 Transcriptional Repressor Function Requires HDAC Activity in the Developing Vertebrate CNS

    PubMed Central

    Wang, Hui; Matise, Michael P.

    2016-01-01

    The generation of functionally distinct neuronal subtypes within the vertebrate central nervous system (CNS) requires the precise regulation of progenitor gene expression in specific neuronal territories during early embryogenesis. Accumulating evidence has implicated histone deacetylase (HDAC) proteins in cell specification, proliferation, and differentiation in diverse embryonic and adult tissues. However, although HDAC proteins have shown to be expressed in the developing vertebrate neural tube, their specific role in CNS neural progenitor fate specification remains unclear. Prior work from our lab showed that the Tcf7l2/Tcf4 transcription factor plays a key role in ventral progenitor lineage segregation by differential repression of two key specification factors, Nkx2.2 and Olig2. In this study, we found that administration of HDAC inhibitors (Valproic Acid (VPA), Trichostatin-A (TSA), or sodium butyrate) in chick embryos in ovo disrupted normal progenitor gene segregation in the developing neural tube, indicating that HDAC activity is required for this process. Further, using functional and pharmacological approaches in vivo, we found that HDAC activity is required for the differential repression of Nkx2.2 and Olig2 by Tcf7l2/Tcf4. Finally, using dominant-negative functional assays, we provide evidence that Tcf7l2/Tcf4 repression also requires Gro/TLE/Grg co-repressor factors. Together, our data support a model where the transcriptional repressor activity of Tcf7l2/Tcf4 involves functional interactions with both HDAC and Gro/TLE/Grg co-factors at specific target gene regulatory elements in the developing neural tube, and that this activity is required for the proper segregation of the Nkx2.2 (p3) and Olig2 (pMN) expressing cells from a common progenitor pool. PMID:27668865

  17. Role of Bound Zn(II) in the CadC Cd(II)/Pb(II)/Zn(II)-Responsive Repressor

    SciTech Connect

    Kandegedara, A.; Thiyagarajan, S; Kondapalli, K; Stemmler, T; Rosen, B

    2009-01-01

    The Staphylococcus aureus plasmid pI258 cadCA operon encodes a P-type ATPase, CadA, that confers resistance to Cd(II)/Pb(II)/Zn(II). Expression is regulated by CadC, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of Cd(II), Pb(II), or Zn(II). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins. The crystal structure of CadC shows two types of metal binding sites, termed Site 1 and Site 2, and the homodimer has two of each. Site 1 is the physiological inducer binding site. The two Site 2 metal binding sites are formed at the dimerization interface. Site 2 is not regulatory in CadC but is regulatory in the homologue SmtB. Here the role of each site was investigated by mutagenesis. Both sites bind either Cd(II) or Zn(II). However, Site 1 has higher affinity for Cd(II) over Zn(II), and Site 2 prefers Zn(II) over Cd(II). Site 2 is not required for either derepression or dimerization. The crystal structure of the wild type with bound Zn(II) and of a mutant lacking Site 2 was compared with the SmtB structure with and without bound Zn(II). We propose that an arginine residue allows for Zn(II) regulation in SmtB and, conversely, a glycine results in a lack of regulation by Zn(II) in CadC. We propose that a glycine residue was ancestral whether the repressor binds Zn(II) at a Site 2 like CadC or has no Site 2 like the paralogous ArsR and implies that acquisition of regulatory ability in SmtB was a more recent evolutionary event.

  18. Knockdown of a Zebrafish Aryl Hydrocarbon Receptor Repressor (AHRRa) Affects Expression of Genes Related to Photoreceptor Development and Hematopoiesis

    PubMed Central

    Aluru, Neelakanteswar; Jenny, Matthew J.; Hahn, Mark E.

    2014-01-01

    The aryl hydrocarbon receptor repressor (AHRR) is a transcriptional repressor of aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF) and is regulated by an AHR-dependent mechanism. Zebrafish (Danio rerio) possess two AHRR paralogs; AHRRa regulates constitutive AHR signaling during development, whereas AHRRb regulates polyaromatic hydrocarbon-induced gene expression. However, little is known about the endogenous roles and targets of AHRRs. The objective of this study was to elucidate the role of AHRRs during zebrafish development using a loss-of-function approach followed by gene expression analysis. Zebrafish embryos were microinjected with morpholino oligonucleotides against AHRRa or AHRRb to knockdown AHRR protein expression. At 72 h postfertilization (hpf), microarray analysis revealed that the expression of 279 and 116 genes was altered by knockdown of AHRRa and AHRRb, respectively. In AHRRa-morphant embryos, 97 genes were up-regulated and 182 genes were down-regulated. Among the down-regulated genes were several related to photoreceptor function, including cone-specific genes such as several opsins (opn1sw1, opn1sw2, opn1mw1, and opn1lw2), phosphodiesterases (pde6H and pde6C), retinol binding protein (rbp4l), phosducin, and arrestins. Down-regulation was confirmed by RT-PCR and with samples from an independent experiment. The four genes tested (opn1sw1, pde6H, pde6C, and arr3b) were not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. AHRRa knockdown also caused up-regulation of embryonic hemoglobin (hbbe3), suggesting a role for AHRR in regulating hematopoiesis. Knockdown of AHRRb caused up-regulation of 31 genes and down-regulation of 85 genes, without enrichment for any specific biological process. Overall, these results suggest that AHRRs may have important roles in development, in addition to their roles in regulating xenobiotic signaling. PMID:24675095

  19. Mouse cytomegalovirus immediate-early protein 1 binds with host cell repressors to relieve suppressive effects on viral transcription and replication during lytic infection.

    PubMed

    Tang, Qiyi; Maul, Gerd G

    2003-01-01

    Herpesviruses start their transcriptional cascade at nuclear domain 10 (ND10). The deposition of virus genomes at these nuclear sites occurs due to the binding of the interferon-inducible repressor protein promyelocytic leukemia protein (PML) and/or Daxx to a viral DNA-protein complex. However, the presence of repressive proteins at the nuclear site of virus transcription has remained unexplained. We investigated the mouse cytomegalovirus (MCMV) immediate-early 1 protein (IE1), which is necessary for productive infection at low multiplicities of infection and therefore likely to be involved in overcoming cellular repression. Temporal analysis of IE1 distribution revealed its initial segregation into ND10 by binding to PML and/or Daxx and IE1-dependent recruitment of the transcriptional repressor histone deacetylase-2 (HDAC-2) to this site. However, these protein aggregates are dissociated in cells producing sufficient IE1 through titration of PML, Daxx, and HDAC-2. Importantly, binding of IE1 to HDAC-2 decreased deacetylation activity. Moreover, inhibition of HDAC by trichostatin-A resulted in an increase in viral protein synthesis, an increase in cells starting the formation of prereplication compartments, and an increase in the total infectious viruses produced. Thus, IE1, like trichostatin-A, reverses the repressive effect of HDAC evident in the presence of acetylated histones in the immediate-early promoter region. Since HDAC also binds to the promoter region of IE1, as determined by the chromatin immunoprecipitation assay, these combined results suggest that IE1 inhibits or reverses HDAC-mediated repression of the infecting viral genomes, possibly by a process akin to activation of heterochromatin. We propose that even permissive cells can repress transcription of infecting viral genomes through repressors, including HDAC, Daxx, and PML, and the segregation of IE1 to ND10 that would inactivate those repressors. The virus can counter this repression by

  20. Adhesin competence repressor (AdcR) from Streptococcus pyogenes controls adaptive responses to zinc limitation and contributes to virulence

    PubMed Central

    Sanson, Misu; Makthal, Nishanth; Flores, Anthony R.; Olsen, Randall J.; Musser, James M.; Kumaraswami, Muthiah

    2015-01-01

    Altering zinc bioavailability to bacterial pathogens is a key component of host innate immunity. Thus, the ability to sense and adapt to the alterations in zinc concentrations is critical for bacterial survival and pathogenesis. To understand the adaptive responses of group A Streptococcus (GAS) to zinc limitation and its regulation by AdcR, we characterized gene regulation by AdcR. AdcR regulates the expression of 70 genes involved in zinc acquisition and virulence. Zinc-bound AdcR interacts with operator sequences in the negatively regulated promoters and mediates differential regulation of target genes in response to zinc deficiency. Genes involved in zinc mobilization and conservation are derepressed during mild zinc deficiency, whereas the energy-dependent zinc importers are upregulated during severe zinc deficiency. Further, we demonstrated that transcription activation by AdcR occurs by direct binding to the promoter. However, the repression and activation by AdcR is mediated by its interactions with two distinct operator sequences. Finally, mutational analysis of the metal ligands of AdcR caused impaired DNA binding and attenuated virulence, indicating that zinc sensing by AdcR is critical for GAS pathogenesis. Together, we demonstrate that AdcR regulates GAS adaptive responses to zinc limitation and identify molecular components required for GAS survival during zinc deficiency. PMID:25510500

  1. Multisite Phosphorylation of the Sum1 Transcriptional Repressor by S-Phase Kinases Controls Exit from Meiotic Prophase in Yeast

    PubMed Central

    Corbi, Daniel; Sunder, Sham; Weinreich, Michael; Skokotas, Aikaterini; Johnson, Erica S.

    2014-01-01

    Activation of the meiotic transcription factor Ndt80 is a key regulatory transition in the life cycle of Saccharomyces cerevisiae because it triggers exit from pachytene and entry into meiosis. The NDT80 promoter is held inactive by a complex containing the DNA-binding protein Sum1 and the histone deacetylase Hst1. Meiosis-specific phosphorylation of Sum1 by the protein kinases Cdk1, Ime2, and Cdc7 is required for NDT80 expression. Here, we show that the S-phase-promoting cyclin Clb5 activates Cdk1 to phosphorylate most, and perhaps all, of the 11 minimal cyclin-dependent kinase (CDK) phospho-consensus sites (S/T-P) in Sum1. Nine of these sites can individually promote modest levels of meiosis, yet these sites function in a quasiadditive manner to promote substantial levels of meiosis. Two Cdk1 sites and an Ime2 site individually promote high levels of meiosis, likely by preparing Sum1 for phosphorylation by Cdc7. Chromatin immunoprecipitation reveals that the phosphorylation sites are required for removal of Sum1 from the NDT80 promoter. We also find that Sum1, but not its partner protein Hst1, is required to repress NDT80 transcription. Thus, while the phosphorylation of Sum1 may lead to dissociation from DNA by influencing Hst1, it is the presence of Sum1 on DNA that determines whether NDT80 will be expressed. PMID:24710277

  2. Multisite phosphorylation of the Sum1 transcriptional repressor by S-phase kinases controls exit from meiotic prophase in yeast.

    PubMed

    Corbi, Daniel; Sunder, Sham; Weinreich, Michael; Skokotas, Aikaterini; Johnson, Erica S; Winter, Edward

    2014-06-01

    Activation of the meiotic transcription factor Ndt80 is a key regulatory transition in the life cycle of Saccharomyces cerevisiae because it triggers exit from pachytene and entry into meiosis. The NDT80 promoter is held inactive by a complex containing the DNA-binding protein Sum1 and the histone deacetylase Hst1. Meiosis-specific phosphorylation of Sum1 by the protein kinases Cdk1, Ime2, and Cdc7 is required for NDT80 expression. Here, we show that the S-phase-promoting cyclin Clb5 activates Cdk1 to phosphorylate most, and perhaps all, of the 11 minimal cyclin-dependent kinase (CDK) phospho-consensus sites (S/T-P) in Sum1. Nine of these sites can individually promote modest levels of meiosis, yet these sites function in a quasiadditive manner to promote substantial levels of meiosis. Two Cdk1 sites and an Ime2 site individually promote high levels of meiosis, likely by preparing Sum1 for phosphorylation by Cdc7. Chromatin immunoprecipitation reveals that the phosphorylation sites are required for removal of Sum1 from the NDT80 promoter. We also find that Sum1, but not its partner protein Hst1, is required to repress NDT80 transcription. Thus, while the phosphorylation of Sum1 may lead to dissociation from DNA by influencing Hst1, it is the presence of Sum1 on DNA that determines whether NDT80 will be expressed.

  3. Decreased expression of Freud-1/CC2D1A, a transcriptional repressor of the 5-HT1A receptor, in the prefrontal cortex of subjects with major depression.

    PubMed

    Szewczyk, Bernadeta; Albert, Paul R; Rogaeva, Anastasia; Fitzgibbon, Heidi; May, Warren L; Rajkowska, Grazyna; Miguel-Hidalgo, Jose J; Stockmeier, Craig A; Woolverton, William L; Kyle, Patrick B; Wang, Zhixia; Austin, Mark C

    2010-09-01

    Serotonin1A (5-HT(1A)) receptors are reported altered in the brain of subjects with major depressive disorder (MDD). Recent studies have identified transcriptional regulators of the 5-HT(1A) receptor and have documented gender-specific alterations in 5-HT(1A) transcription factor and 5-HT(1A) receptors in female MDD subjects. The 5' repressor element under dual repression binding protein-1 (Freud-1) is a calcium-regulated repressor that negatively regulates the 5-HT(1A) receptor gene. This study documented the cellular expression of Freud-1 in the human prefrontal cortex (PFC) and quantified Freud-1 protein in the PFC of MDD and control subjects as well as in the PFC of rhesus monkeys chronically treated with fluoxetine. Freud-1 immunoreactivity was present in neurons and glia and was co-localized with 5-HT(1A) receptors. Freud-1 protein level was significantly decreased in the PFC of male MDD subjects (37%, p=0.02) relative to gender-matched control subjects. Freud-1 protein was also reduced in the PFC of female MDD subjects (36%, p=0.18) but was not statistically significant. When the data was combined across genders and analysed by age, the decrease in Freud-1 protein level was greater in the younger MDD subjects (48%, p=0.01) relative to age-matched controls as opposed to older depressed subjects. Similarly, 5-HT(1A) receptor protein was significantly reduced in the PFC of the younger MDD subjects (48%, p=0.01) relative to age-matched controls. Adult male rhesus monkeys administered fluoxetine daily for 39 wk revealed no significant change in cortical Freud-1 or 5-HT(1A) receptor proteins compared to vehicle-treated control monkeys. Reduced protein expression of Freud-1 in MDD subjects may reflect dysregulation of this transcription factor, which may contribute to the altered regulation of 5-HT(1A) receptors observed in subjects with MDD. These data may also suggest that reductions in Freud-1 protein expression in the PFC may be associated with early onset of

  4. Methicillin-Resistance in Staphylococcus aureus Is Not Affected by the Overexpression in Trans of the mecA Gene Repressor: A Surprising Observation

    PubMed Central

    Oliveira, Duarte C.; de Lencastre, Hermínia

    2011-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is intrinsically cross-resistant to virtually all β-lactam antibiotics. The central determinant for the MRSA phenotype is the mecA gene, whose transcriptional control may be mediated by a repressor (mecI) and a sensor/inducer (mecR1). The mecI-mecR1-mediated induction of mecA takes several hours rendering the strains phenotypically susceptible in spite of the presence of the resistance gene. Therefore, it has been proposed that the full resistance to β-lactams observed in many contemporary clinical MRSA strains requires a non-functional mecI-mecR1 regulatory system. The mecA gene is embedded in a large chromosomal cassette (the SCCmec element) for which several structural types have been described. Some epidemic MRSA clones, typically expressing full β-lactam resistance, carry SCCmec elements that contain an intact mecI-mecR1 locus (e.g. SCCmec types II and III). We have addressed this apparent contradiction by first sequencing the mecI coding region and mecA promoter sequences in a collection of prototype MRSA strains characterized by different SCCmec types. A conserved non-sense mutation within mecI was detected in all SCCmec type III strains tested, presumably responsible for a non-functional truncated MecI protein and, therefore, explaining the full resistance phenotype. In SCCmec type II strains no conserved mutations were found. We next transformed a collection of prototype MRSA epidemic strains with a recombinant plasmid overexpressing a wild-type copy of mecI. Surprisingly, for the great majority of the strains no significant alterations in the phenotypic expression of β-lactam resistance could be detected. These findings were confirmed and further explored, challenging the currently accepted mechanism of mecA transcriptional control. Our observations suggest the existence of yet unidentified additional determinants involved in the transcriptional control of mecA gene and point to a revision of the mec

  5. Expression, Purification And Preliminary X-Ray Analysis of the C-Terminal Domain of An Arginine Repressor Protein From Mycobacterium Tuberculosis

    SciTech Connect

    Lu, G.J.; Garen, C.R.; Cherney, M.M.; Cherney, L.T.; Lee, C.; James, M.N.J.

    2009-06-03

    The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 {angstrom}. The crystals belong to space group P1 and the Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of six molecules per unit cell.

  6. The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site.

    PubMed

    Hammerl, Jens Andre; Roschanski, Nicole; Lurz, Rudi; Johne, Reimar; Lanka, Erich; Hertwig, Stefan

    2015-06-01

    Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O\\(_{\\rm{R}}\\)3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O\\(_{\\rm{R}}\\)3) upstream of the prophage repressor gene. The O\\(_{\\rm{R}}\\)3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages. PMID:26043380

  7. Involvement of co-repressor LUH and the adapter proteins SLK1 and SLK2 in the regulation of abiotic stress response genes in Arabidopsis

    PubMed Central

    2014-01-01

    Background During abiotic stress many genes that are important for growth and adaptation to stress are expressed at elevated levels. However, the mechanisms that keep the stress responsive genes from expressing under non stress conditions remain elusive. Recent genetic characterization of the co-repressor LEUNIG_HOMOLOG (LUH) and transcriptional adaptor proteins SEUSS-LIKE1 (SLK1) and SLK2 have been proposed to function redundantly in diverse developmental processes; however their function in the abiotic stress response is unknown. Moreover, the molecular functions of LUH, SLK1 and SLK2 remain obscure. Here, we show the molecular function of LUH, SLK1 and SLK2 and the role of this complex in the abiotic stress response. Results The luh, slk1 and slk2 mutant plants shows enhanced tolerance to salt and osmotic stress conditions. SLK1 and SLK2 interact physically with the LUFS domain in LUH forming SLK1-LUH and SLK2-LUH co-repressor complexes to inhibit the transcription. LUH has repressor activity, whereas SLK1 and SLK2 function as adaptors to recruit LUH, which in turn recruits histone deacetylase to the target sequences to repress transcription. The stress response genes RD20, MYB2 and NAC019 are expressed at elevated levels in the luh, slk1 and slk2 mutant plants. Furthermore, these stress response genes are associated with decreased nucleosome density and increased acetylation levels at H3K9 and H3K14 in the luh, slk1 and slk2 mutant plants. Conclusions Our results indicate that SLK1, SLK2 and LUH form a co-repressor complex. LUH represses by means of an epigenetic process involving histone modification to facilitate the condensation of chromatin thus preventing transcription at the target genes. PMID:24564815

  8. Robust specification of sensory neurons by dual functions of charlatan, a Drosophila NRSF/REST-like repressor of extramacrochaetae and hairy.

    PubMed

    Yamasaki, Yasutoyo; Lim, Young-Mi; Niwa, Nao; Hayashi, Shigeo; Tsuda, Leo

    2011-08-01

    Sensory bristle formation in Drosophila is a well-characterized system for studying sensory organ development at the molecular level. The master proneural genes of the achaete-scute (ac-sc) complex, which encode basic-helix-loop-helix (bHLH) transcription factors, are necessary and sufficient for sensory bristle formation. charlatan (chn) was originally identified as a transcriptional activator of ac-sc gene expression through interaction with its enhancer, an activity that promotes sensory bristle development. In contrast, Chn was also identified as a functional homologue of mammalian neuron-restrictive silencing factor or RE1 silencing transcription factor (NRSF/REST), an important transcriptional repressor during vertebrate neurogenesis and stem cell development that acts through epigenetic gene silencing. Here, we report that Chn acts as a repressor of extramacrochaetae (emc) and hairy, molecules that inhibit ac-sc expression. This double-negative mechanism, together with direct activation via the achaete enhancer, increases expression of achaete and ensures robust development of sensory neurons. A mutation in the C-terminal repressor motif of Chn, which causes Chn to lose its repression activity, converted Chn to an activator of emc and hairy, suggesting that Chn is a dual functional regulator of transcription. Because chn-like sequences are found among arthropods, regulation of neuronal development by Chn-like molecules may be widely conserved. PMID:21762412

  9. Short linear motif acquisition, exon formation and alternative splicing determine a pathway to diversity for NCoR-family co-repressors

    PubMed Central

    Short, Stephen; Peterkin, Tessa; Guille, Matthew; Patient, Roger; Sharpe, Colin

    2015-01-01

    Vertebrate NCoR-family co-repressors play central roles in the timing of embryo and stem cell differentiation by repressing the activity of a range of transcription factors. They interact with nuclear receptors using short linear motifs (SLiMs) termed co-repressor for nuclear receptor (CoRNR) boxes. Here, we identify the pathway leading to increasing co-repressor diversity across the deuterostomes. The final complement of CoRNR boxes arose in an ancestral cephalochordate, and was encoded in one large exon; the urochordates and vertebrates then split this region between 10 and 12 exons. In Xenopus, alternative splicing is prevalent in NCoR2, but absent in NCoR1. We show for one NCoR1 exon that alternative splicing can be recovered by a single point mutation, suggesting NCoR1 lost the capacity for alternative splicing. Analyses in Xenopus and zebrafish identify that cellular context, rather than gene sequence, predominantly determines species differences in alternative splicing. We identify a pathway to diversity for the NCoR family beginning with the addition of a SLiM, followed by gene duplication, the generation of alternatively spliced isoforms and their differential deployment. PMID:26289800

  10. Studies on the interaction of REST4 with the cholinergic repressor element-1/neuron restrictive silencer element.

    PubMed

    Lee, J H; Shimojo, M; Chai, Y G; Hersh, L B

    2000-08-14

    REST4 is a neuron specific truncated form of the transcription factor REST/NRSE derived by alternative splicing. REST4 was previously shown to block the repressor activity of REST/NRSF by forming a hetero-oligomer, Shimojo et al. [Mol. Cell. Biol. 19 (1999) 6788-6795]. A series of deletion mutants have now been used to characterize REST4 in terms of its structure and DNA binding. REST4 was found to be O-glycosylated between between residues 87 and 152. Binding of REST4 to the cholinergic RE-1/NRSE was approximately 1/10 to 1/20 as strong as full length REST/NRSF. DNA binding was enhanced by deletion of the first 86 residues and was found to require all four of the C-terminal zinc fingers as well as a twelve amino acid sequence preceding the first of these zinc fingers. REST4 can form homo-oligomers, however only the monomer was found to bind to DNA. REST4 binds to the 3' sequence of the cholinergic NRSE suggesting an anti-parallel orientation of the protein to the DNA. PMID:11039732

  11. Activation of the Nrf2 pathway by inorganic arsenic in human hepatocytes and the role of transcriptional repressor Bach1.

    PubMed

    Liu, Dan; Duan, Xiaoxu; Dong, Dandan; Bai, Caijun; Li, Xin; Sun, Guifan; Li, Bing

    2013-01-01

    Previous studies have proved that the environmental toxicant, inorganic arsenic, activates nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in many different cell types. This study tried to explore the hepatic Nrf2 pathway upon arsenic treatment comprehensively, since liver is one of the major target organs of arsenical toxicity. Our results showed that inorganic arsenic significantly induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also observed a dose-dependent increase of antioxidant response element- (ARE-) luciferase activity. Both the mRNA and protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) were all upregulated dramatically. On the other hand, entry and accumulation of Nrf2 protein in the nucleus, while exportting the transcriptional repressor BTB and CNC homology 1 (Bach1) from nucleus to cytoplasm, were also confirmed by western blot and immunofluorescence assay. Our results therefore confirmed the arsenic-induced Nrf2 pathway activation in hepatocytes and also suggested that the translocation of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic Nrf2 pathway plays indispensable roles for cellular defenses against arsenic hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand the self-defensive responses and the diverse biological effects of arsenicals.

  12. Functional impact of polar and acidic substitutions in the lactose repressor hydrophobic monomer.monomer interface with a buried lysine.

    PubMed

    Zhan, Hongli; Sun, Zhifei; Matthews, Kathleen Shive

    2009-02-17

    Despite predicted energetic penalties, the charged K84 side chains of tetrameric lactose repressor protein (LacI) are found buried within the highly hydrophobic monomer.monomer interface that includes side chains of V94 and V96. Once inducer binding has occurred, these K84 side chains move to interact with the more solvent-exposed side chains of D88 and E100'. Previous studies demonstrated that hydrophobic substitutions for K84 increased protein stability and significantly impaired the allosteric response. These results indicated that enhanced hydrophobic interactions at the monomer.monomer interface remove the energetic driving force of the buried charges, decreasing the likelihood of a robust conformational change and stabilizing the structure. We hypothesized that creating a salt bridge network with the lysine side chains by including nearby negatively charged residues might result in a similar outcome. To that end, acidic residues, D and E, and their neutral amides, N and Q, were substituted for the valines at positions 94 and 96. These variants exhibited one or more of the following functional changes: weakened inducer binding, impaired allosteric response, and diminished protein stability. For V96D and V96E, ion pair formation with K84 appears optimal, and the loss of inducer response exceeds that of the hydrophobic K84A and -L variants. However, impacts on functional properties indicate that stabilizing the buried positive charge with polar or ion pair interactions is not functionally equivalent to structural stabilization via hydrophobic enhancement. PMID:19166325

  13. The experimental folding landscape of monomeric lactose repressor, a large two-domain protein, involves two kinetic intermediates.

    PubMed

    Wilson, Corey J; Das, Payel; Clementi, Cecilia; Matthews, Kathleen S; Wittung-Stafshede, Pernilla

    2005-10-11

    To probe the experimental folding behavior of a large protein with complex topology, we created a monomeric variant of the lactose repressor protein (MLAc), a well characterized tetrameric protein that regulates transcription of the lac operon. Purified MLAc is folded, fully functional, and binds the inducer isopropyl beta-d-thiogalactoside with the same affinity as wild-type LacI. Equilibrium unfolding of MLAc induced by the chemical denaturant urea is a reversible, apparent two-state process (pH 7.5, 20 degrees C). However, time-resolved experiments demonstrate that unfolding is single-exponential, whereas refolding data indicate two transient intermediates. The data reveal the initial formation of a burst-phase (tau < ms) intermediate that corresponds to approximately 50% of the total secondary-structure content. This step is followed by a rearrangement reaction that is rate-limited by an unfolding process (tau approximately 3 s; pH 7.5, 20 degrees C) and results in a second intermediate. This MLAc intermediate converts to the native structure (tau approximately 30 s; pH 7.5, 20 degrees C). Remarkably, the experimental folding-energy landscape for MLAc is in excellent agreement with theoretical predictions using a simple topology-based C(alpha)-model as presented in a companion article in this issue. PMID:16203983

  14. Enhanced bacterial protein expression during auto-induction obtained by alteration of lac repressor dosage and medium composition.

    PubMed

    Blommel, Paul G; Becker, Katie J; Duvnjak, Petar; Fox, Brian G

    2007-01-01

    The auto-induction method of protein expression in E. coli is based on diauxic growth resulting from dynamic function of lac operon regulatory elements (lacO and LacI) in mixtures of glucose, glycerol, and lactose. The results show that successful execution of auto-induction is strongly dependent on the plasmid promoter and repressor construction, on the oxygenation state of the culture, and on the composition of the auto-induction medium. Thus expression hosts expressing high levels of LacI during aerobic growth exhibit reduced ability to effectively complete the auto-induction process. Manipulation of the promoter to decrease the expression of LacI altered the preference for lactose consumption in a manner that led to increased protein expression and partially relieved the sensitivity of the auto-induction process to the oxygenation state of the culture. Factorial design methods were used to optimize the chemically defined growth medium used for expression of two model proteins, Photinus luciferase and enhanced green fluorescent protein, including variations for production of both unlabeled and selenomethionine-labeled samples. The optimization included studies of the expression from T7 and T7-lacI promoter plasmids and from T5 phage promoter plasmids expressing two levels of LacI. Upon the basis of the analysis of over 500 independent expression results, combinations of optimized expression media and expression plasmids that gave protein yields of greater than 1000 mug/mL of expression culture were identified. PMID:17506520

  15. Sliding and target location of DNA-binding proteins: an NMR view of the lac repressor system.

    PubMed

    Loth, Karine; Gnida, Manuel; Romanuka, Julija; Kaptein, Robert; Boelens, Rolf

    2013-05-01

    In non-specific lac headpiece-DNA complexes selective NMR line broadening is observed that strongly depends on length and composition of the DNA fragments. This broadening involves amide protons found in the non-specific lac-DNA structure to be interacting with the DNA phosphate backbone, and can be ascribed to DNA sliding of the protein along the DNA. This NMR exchange broadening has been used to estimate the 1D diffusion constant for sliding along non-specific DNA. The observed 1D diffusion constant of 4×10(-12) cm(2)/s is two orders of magnitude smaller than derived from previous kinetic experiments, but falls in the range of values determined more recently using single molecule methods. This strongly supports the notion that sliding could play at most a minor role in the association kinetics of binding of lac repressor to lac operator and that other processes such as hopping and intersegment transfer contribute to facilitate the DNA recognition process.

  16. Inactivation of Burkholderia cepacia Complex Phage KS9 gp41 Identifies the Phage Repressor and Generates Lytic Virions▿ †

    PubMed Central

    Lynch, Karlene H.; Seed, Kimberley D.; Stothard, Paul; Dennis, Jonathan J.

    2010-01-01

    The Burkholderia cepacia complex (BCC) is made up of at least 17 species of Gram-negative opportunistic bacterial pathogens that cause fatal infections in patients with cystic fibrosis and chronic granulomatous disease. KS9 (vB_BcenS_KS9), one of a number of temperate phages isolated from BCC species, is a prophage of Burkholderia pyrrocinia LMG 21824. Transmission electron micrographs indicate that KS9 belongs to the family Siphoviridae and exhibits the B1 morphotype. The 39,896-bp KS9 genome, comprised of 50 predicted genes, integrates into the 3′ end of the LMG 21824 GTP cyclohydrolase II open reading frame. The KS9 genome is most similar to uncharacterized prophage elements in the genome of B. cenocepacia PC184 (vB_BcenZ_ PC184), as well as Burkholderia thailandensis phage φE125 and Burkholderia pseudomallei phage φ1026b. Using molecular techniques, we have disrupted KS9 gene 41, which exhibits similarity to genes encoding phage repressors, producing a lytic mutant named KS9c. This phage is incapable of stable lysogeny in either LMG 21824 or B. cenocepacia strain K56-2 and rescues a Galleria mellonella infection model from experimental B. cenocepacia K56-2 infections at relatively low multiplicities of infection. These results readily demonstrate that temperate phages can be genetically engineered to lytic form and that these modified phages can be used to treat bacterial infections in vivo. PMID:19939932

  17. Yap7 is a transcriptional repressor of nitric oxide oxidase in yeasts, which arose from neofunctionalization after whole genome duplication.

    PubMed

    Merhej, Jawad; Delaveau, Thierry; Guitard, Juliette; Palancade, Benoit; Hennequin, Christophe; Garcia, Mathilde; Lelandais, Gaëlle; Devaux, Frédéric

    2015-06-01

    Flavohemoglobins are the main detoxifiers of nitric oxide (NO) in bacteria and fungi and are induced in response to nitrosative stress. In fungi, the flavohemoglobin encoding gene YHB1 is positively regulated by transcription factors which are activated upon NO exposure. In this study, we show that in the model yeast Saccharomyces cerevisiae and in the human pathogen Candida glabrata, the transcription factor Yap7 constitutively represses YHB1 by binding its promoter. Consequently, YAP7 deletion conferred high NO resistance to the cells. Co-immunoprecipitation experiments and mutant analyses indicated that Yap7 represses YHB1 by recruiting the transcriptional repressor Tup1. In S. cerevisiae, YHB1 repression also involves interaction of Yap7 with the Hap2/3/5 complex through a conserved Hap4-like-bZIP domain, but this interaction has been lost in C. glabrata. The evolutionary origin of this regulation was investigated by functional analyses of Yap7 and of its paralogue Yap5 in different yeast species. These analyses indicated that the negative regulation of YHB1 by Yap7 arose by neofunctionalization after the whole genome duplication which led to the C. glabrata and S. cerevisiae extant species. This work describes a new aspect of the regulation of fungal nitric oxidase and provides detailed insights into its functioning and evolution.

  18. The Transcriptional Repressor MYB2 Regulates Both Spatial and Temporal Patterns of Proanthocyandin and Anthocyanin Pigmentation in Medicago truncatula.

    PubMed

    Jun, Ji Hyung; Liu, Chenggang; Xiao, Xirong; Dixon, Richard A

    2015-10-01

    Accumulation of anthocyanins and proanthocyanidins (PAs) is limited to specific cell types and developmental stages, but little is known about how antagonistically acting transcriptional regulators work together to determine temporal and spatial patterning of pigmentation at the cellular level, especially for PAs. Here, we characterize MYB2, a transcriptional repressor regulating both anthocyanin and PA biosynthesis in the model legume Medicago truncatula. MYB2 was strongly upregulated by MYB5, a major regulator of PA biosynthesis in M. truncatula and a component of MYB-basic helix loop helix-WD40 (MBW) activator complexes. Overexpression of MYB2 abolished anthocyanin and PA accumulation in M. truncatula hairy roots and Arabidopsis thaliana seeds, respectively. Anthocyanin deposition was expanded in myb2 mutant seedlings and flowers accompanied by increased anthocyanin content. PA mainly accumulated in the epidermal layer derived from the outer integument in the M. truncatula seed coat, starting from the hilum area. The area of PA accumulation and ANTHOCYANIDIN REDUCTASE expression was expanded into the seed body at the early stage of seed development in the myb2 mutant. Genetic, biochemical, and cell biological evidence suggests that MYB2 functions as part of a multidimensional regulatory network to define the temporal and spatial pattern of anthocyanin and PA accumulation linked to developmental processes.

  19. T Cell Fates Zipped Up: How the Bach2 Basic Leucine Zipper Transcriptional Repressor Directs T Cell Differentiation and Function.

    PubMed

    Richer, Martin J; Lang, Mark L; Butler, Noah S

    2016-08-15

    Recent data illustrate a key role for the transcriptional regulator bric-a-brac, tramtrack, and broad complex and cap'n'collar homology (Bach)2 in orchestrating T cell differentiation and function. Although Bach2 has a well-described role in B cell differentiation, emerging data show that Bach2 is a prototypical member of a novel class of transcription factors that regulates transcriptional activity in T cells at super-enhancers, or regions of high transcriptional activity. Accumulating data demonstrate specific roles for Bach2 in favoring regulatory T cell generation, restraining effector T cell differentiation, and potentiating memory T cell development. Evidence suggests that Bach2 regulates various facets of T cell function by repressing other key transcriptional regulators such as B lymphocyte-induced maturation protein 1. In this review, we examine our present understanding of the role of Bach2 in T cell function and highlight the growing evidence that this transcriptional repressor functions as a key regulator involved in maintenance of T cell quiescence, T cell subset differentiation, and memory T cell generation.

  20. Deficiency of the transcriptional repressor B cell lymphoma 6 (Bcl6) is accompanied by dysregulated lipid metabolism.

    PubMed

    LaPensee, Christopher R; Lin, Grace; Dent, Alexander L; Schwartz, Jessica

    2014-01-01

    The transcriptional repressor B-cell Lymphoma 6 (Bcl6) was recently identified in a profile of genes regulated in adipocytes, suggesting a relationship between Bcl6 and metabolic regulation. As a representative target gene repressed by Bcl6, Suppressor of Cytokine Signaling (Socs) 2 expression was elevated in Bcl6 deficient (KO) mice, including metabolic tissues liver, adipose tissue and muscle, as well as in spleen and thymus. Bcl6 occupied the Socs2 promoter in wild-type, but not Bcl6 KO mice, suggesting direct regulation of Socs2 by Bcl6 in vivo. Mice deficient in Bcl6 were found to exhibit multiple features of dysregulated lipid metabolism. Adipose tissue mass was dramatically reduced or absent in Bcl6 KO mice. Further, hepatic and serum triglycerides were low. Bcl6 deficiency was accompanied by decreased hepatic expression of Stearoyl-CoA desaturase 1 (Scd1) and Fatty acid synthase (Fasn) genes which encode lipogenic enzymes. Expression of the gene for the transcription factor Carbohydrate-Responsive Element Binding Protein (Chrebp), which regulates expression of lipogenic genes, was also reduced in liver of Bcl6 KO mice. Bcl6 deficiency disrupted fasting-induced increases in hepatic triglyceride deposition, but not decreases in lipogenic gene expression. Taken together, these findings suggest that in addition to its well-recognized roles in immune regulation, Bcl6 plays a role in regulatory events of lipid metabolism, and that in the absence of Bcl6, lipid metabolism in liver and adipose tissue is dysregulated.

  1. The Transcriptional Repressor Polycomb Group Factor 6, PCGF6, Negatively Regulates Dendritic Cell Activation and Promotes Quiescence.

    PubMed

    Boukhaled, Giselle M; Cordeiro, Brendan; Deblois, Genevieve; Dimitrov, Vassil; Bailey, Swneke D; Holowka, Thomas; Domi, Anisa; Guak, Hannah; Chiu, Huai-Hsuan Clare; Everts, Bart; Pearce, Edward J; Lupien, Mathieu; White, John H; Krawczyk, Connie M

    2016-08-16

    Pro-inflammatory signals provided by the microenvironment are critical to activate dendritic cells (DCs), components of the innate immune system that shape both innate and adaptive immunity. However, to prevent inappropriate immune activation, mechanisms must be in place to restrain DC activation to ensure DCs are activated only once sufficient stimuli have been received. Here, we report that DC activation and immunogenicity are regulated by the transcriptional repressor Polycomb group factor 6 (PCGF6). Pcgf6 is rapidly downregulated upon stimulation, and this downregulation is necessary to permit full DC activation. Silencing PCGF6 expression enhanced both spontaneous and stimulated DC activation. We show that PCGF6 associates with the H3K4me3 demethylase JARID1c, and together, they negatively regulate H3K4me3 levels in DCs. Our results identify two key regulators, PCGF6 and JARID1c that temper DC activation and implicate active transcriptional silencing via histone demethylation as a previously unappreciated mechanism for regulating DC activation and quiescence.

  2. Activation of the Nrf2 pathway by inorganic arsenic in human hepatocytes and the role of transcriptional repressor Bach1.

    PubMed

    Liu, Dan; Duan, Xiaoxu; Dong, Dandan; Bai, Caijun; Li, Xin; Sun, Guifan; Li, Bing

    2013-01-01

    Previous studies have proved that the environmental toxicant, inorganic arsenic, activates nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in many different cell types. This study tried to explore the hepatic Nrf2 pathway upon arsenic treatment comprehensively, since liver is one of the major target organs of arsenical toxicity. Our results showed that inorganic arsenic significantly induced Nrf2 protein and mRNA expression in Chang human hepatocytes. We also observed a dose-dependent increase of antioxidant response element- (ARE-) luciferase activity. Both the mRNA and protein levels of NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) were all upregulated dramatically. On the other hand, entry and accumulation of Nrf2 protein in the nucleus, while exportting the transcriptional repressor BTB and CNC homology 1 (Bach1) from nucleus to cytoplasm, were also confirmed by western blot and immunofluorescence assay. Our results therefore confirmed the arsenic-induced Nrf2 pathway activation in hepatocytes and also suggested that the translocation of Bach1 was associated with the regulation of Nrf2 pathway by arsenic. Hepatic Nrf2 pathway plays indispensable roles for cellular defenses against arsenic hepatotoxicity, and the interplay of Bach1 and Nrf2 may be helpful to understand the self-defensive responses and the diverse biological effects of arsenicals. PMID:23738048

  3. Crystal structure of caspase recruiting domain (CARD) of apoptosis repressor with CARD (ARC) and its implication in inhibition of apoptosis.

    PubMed

    Jang, Tae-ho; Kim, Seong Hyun; Jeong, Jae-Hee; Kim, Sunghwan; Kim, Yeon-Gil; Kim, Yeun Gil; Park, Hyun Ho

    2015-01-01

    Apoptosis repressor with caspase recruiting domain (ARC) is a multifunctional inhibitor of apoptosis that is unusually over-expressed or activated in various cancers and in the state of the pulmonary hypertension. Therefore, ARC might be an optimal target for therapeutic intervention. Human ARC is composed of two distinct domains, N-terminal caspase recruiting domain (CARD) and C-terminal P/E (proline and glutamic acid) rich domain. ARC inhibits the extrinsic apoptosis pathway by interfering with DISC formation. ARC CARD directly interacts with the death domains (DDs) of Fas and FADD, as well as with the death effector domains (DEDs) of procaspase-8. Here, we report the first crystal structure of the CARD domain of ARC at a resolution of 2.4 Å. Our structure was a dimer with novel homo-dimerization interfaces that might be critical to its inhibitory function. Interestingly, ARC did not exhibit a typical death domain fold. The sixth helix (H6), which was detected at the typical death domain fold, was not detected in the structure of ARC, indicating that H6 may be dispensable for the function of the death domain superfamily.

  4. The BEN domain is a novel sequence-specific DNA-binding domain conserved in neural transcriptional repressors

    PubMed Central

    Dai, Qi; Ren, Aiming; Westholm, Jakub O.; Serganov, Artem A.; Patel, Dinshaw J.; Lai, Eric C.

    2013-01-01

    We recently reported that Drosophila Insensitive (Insv) promotes sensory organ development and has activity as a nuclear corepressor for the Notch transcription factor Suppressor of Hairless [Su(H)]. Insv lacks domains of known biochemical function but contains a single BEN domain (i.e., a “BEN-solo” protein). Our chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) analysis confirmed binding of Insensitive to Su(H) target genes in the Enhancer of split gene complex [E(spl)-C]; however, de novo motif analysis revealed a novel site strongly enriched in Insv peaks (TCYAATHRGAA). We validate binding of endogenous Insv to genomic regions bearing such sites, whose associated genes are enriched for neural functions and are functionally repressed by Insv. Unexpectedly, we found that the Insv BEN domain binds specifically to this sequence motif and that Insv directly regulates transcription via this motif. We determined the crystal structure of the BEN–DNA target complex, revealing homodimeric binding of the BEN domain and extensive nucleotide contacts via α helices and a C-terminal loop. Point mutations in key DNA-contacting residues severely impair DNA binding in vitro and capacity for transcriptional regulation in vivo. We further demonstrate DNA-binding and repression activities by the mammalian neural BEN-solo protein BEND5. Altogether, we define novel DNA-binding activity in a conserved family of transcriptional repressors, opening a molecular window on this extensive gene family. PMID:23468431

  5. Contributions of a hydrogen bond/salt bridge network to the stability of secondary and tertiary structure in lambda repressor.

    PubMed Central

    Marqusee, S.; Sauer, R. T.

    1994-01-01

    In the N-terminal domain of lambda repressor, the Asp 14 side chain forms an intrahelical, hydrogen bond/salt bridge with the Arg 17 side chain and a tertiary hydrogen bond with the Ser 77 side chain. By measuring the stabilities to urea denaturation of the wild-type N-terminal domain and variants containing single, double, and triple alanine substitutions at positions 14, 17, and 77, the side-chain interaction energies, the coupling energy between interactions, and the intrinsic effects of each wild-type side chain on protein stability have been estimated. These studies indicate that the Asp 14-Arg 17 and Asp 14-Ser 77 interactions are stabilizing by roughly 0.8 and 1.5 kcal/mol, respectively, but that Asp 14, by itself, is destabilizing by roughly 0.9 kcal/mol. We also show that a peptide model of alpha-helix 1, which contains Asp 14 and Arg 17, forms a reasonably stable, monomeric helix in solution and responds to alanine mutations at positions 14 and 17 in the fashion expected from the intact protein studies. These studies suggest that it is possible to view the stability effects of mutations in intact proteins in a hierarchical fashion, with the stability of units of secondary structure being distinguishable from the stability of tertiary structure. PMID:7756981

  6. A repressor activator protein1 homologue from an oleaginous strain of Candida tropicalis increases storage lipid production in Saccharomyces cerevisiae.

    PubMed

    Chattopadhyay, Atrayee; Dey, Prabuddha; Barik, Amita; Bahadur, Ranjit P; Maiti, Mrinal K

    2015-06-01

    The repressor activator protein1 (Rap1) has been studied over the years as a multifunctional regulator in Saccharomyces cerevisiae. However, its role in storage lipid accumulation has not been investigated. This report documents the identification and isolation of a putative transcription factor CtRap1 gene from an oleaginous strain of Candida tropicalis, and establishes the direct effect of its expression on the storage lipid accumulation in S. cerevisiae, usually a non-oleaginous yeast. In silico analysis revealed that the CtRap1 polypeptide binds relatively more strongly to the promoter of fatty acid synthase1 (FAS1) gene of S. cerevisiae than ScRap1. The expression level of CtRap1 transcript in vivo was found to correlate directly with the amount of lipid produced in oleaginous native host C. tropicalis. Heterologous expression of the CtRap1 gene resulted in ∼ 4-fold enhancement of storage lipid content (57.3%) in S. cerevisiae. We also showed that the functionally active CtRap1 upregulates the endogenous ScFAS1 and ScDGAT genes of S. cerevisiae, and this, in turn, might be responsible for the increased lipid production in the transformed yeast. Our findings pave the way for the possible utility of the CtRap1 gene in suitable microorganisms to increase their storage lipid content through transcription factor engineering.

  7. Balancing intestinal and systemic inflammation through cell type-specific expression of the aryl hydrocarbon receptor repressor

    PubMed Central

    Brandstätter, Olga; Schanz, Oliver; Vorac, Julia; König, Jessica; Mori, Tetsushi; Maruyama, Toru; Korkowski, Markus; Haarmann-Stemmann, Thomas; von Smolinski, Dorthe; Schultze, Joachim L.; Abel, Josef; Esser, Charlotte; Takeyama, Haruko; Weighardt, Heike; Förster, Irmgard

    2016-01-01

    As a sensor of polyaromatic chemicals the aryl hydrocarbon receptor (AhR) exerts an important role in immune regulation besides its requirement for xenobiotic metabolism. Transcriptional activation of AhR target genes is counterregulated by the AhR repressor (AhRR) but the exact function of the AhRR in vivo is currently unknown. We here show that the AhRR is predominantly expressed in immune cells of the skin and intestine, different from other AhR target genes. Whereas AhRR antagonizes the anti-inflammatory function of the AhR in the context of systemic endotoxin shock, AhR and AhRR act in concert to dampen intestinal inflammation. Specifically, AhRR contributes to the maintenance of colonic intraepithelial lymphocytes and prevents excessive IL-1β production and Th17/Tc17 differentiation. In contrast, the AhRR enhances IFN-γ-production by effector T cells in the inflamed gut. Our findings highlight the physiologic importance of cell-type specific balancing of AhR/AhRR expression in response to microbial, nutritional and other environmental stimuli. PMID:27184933

  8. Balancing intestinal and systemic inflammation through cell type-specific expression of the aryl hydrocarbon receptor repressor.

    PubMed

    Brandstätter, Olga; Schanz, Oliver; Vorac, Julia; König, Jessica; Mori, Tetsushi; Maruyama, Toru; Korkowski, Markus; Haarmann-Stemmann, Thomas; von Smolinski, Dorthe; Schultze, Joachim L; Abel, Josef; Esser, Charlotte; Takeyama, Haruko; Weighardt, Heike; Förster, Irmgard

    2016-05-17

    As a sensor of polyaromatic chemicals the aryl hydrocarbon receptor (AhR) exerts an important role in immune regulation besides its requirement for xenobiotic metabolism. Transcriptional activation of AhR target genes is counterregulated by the AhR repressor (AhRR) but the exact function of the AhRR in vivo is currently unknown. We here show that the AhRR is predominantly expressed in immune cells of the skin and intestine, different from other AhR target genes. Whereas AhRR antagonizes the anti-inflammatory function of the AhR in the context of systemic endotoxin shock, AhR and AhRR act in concert to dampen intestinal inflammation. Specifically, AhRR contributes to the maintenance of colonic intraepithelial lymphocytes and prevents excessive IL-1β production and Th17/Tc17 differentiation. In contrast, the AhRR enhances IFN-γ-production by effector T cells in the inflamed gut. Our findings highlight the physiologic importance of cell-type specific balancing of AhR/AhRR expression in response to microbial, nutritional and other environmental stimuli.

  9. The LSD1 Family of Histone Demethylases and the Pumilio Posttranscriptional Repressor Function in a Complex Regulatory Feedback Loop

    PubMed Central

    Miles, Wayne O.; Lepesant, Julie M. J.; Bourdeaux, Jessie; Texier, Manuela; Kerenyi, Marc A.; Nakakido, Makoto; Hamamoto, Ryuji; Orkin, Stuart H.; Dyson, Nicholas J.

    2015-01-01

    The lysine (K)-specific demethylase (LSD1) family of histone demethylases regulates chromatin structure and the transcriptional potential of genes. LSD1 is frequently deregulated in tumors, and depletion of LSD1 family members causes developmental defects. Here, we report that reductions in the expression of the Pumilio (PUM) translational repressor complex enhanced phenotypes due to dLsd1 depletion in Drosophila. We show that the PUM complex is a target of LSD1 regulation in fly and mammalian cells and that its expression is inversely correlated with LSD1 levels in human bladder carcinoma. Unexpectedly, we find that PUM posttranscriptionally regulates LSD1 family protein levels in flies and human cells, indicating the existence of feedback loops between the LSD1 family and the PUM complex. Our results highlight a new posttranscriptional mechanism regulating LSD1 activity and suggest that the feedback loop between the LSD1 family and the PUM complex may be functionally important during development and in human malignancies. PMID:26438601

  10. Modulation of Ultrafast Conformational Dynamics in Allosteric Interaction of Gal Repressor Protein with Different Operator DNA Sequences.

    PubMed

    Choudhury, Susobhan; Naiya, Gitashri; Singh, Priya; Lemmens, Peter; Roy, Siddhartha; Pal, Samir Kumar

    2016-04-01

    Although all forms of dynamical behaviour of a protein under allosteric interaction with effectors are predicted, little evidence of ultrafast dynamics in the interaction has been reported. Here, we demonstrate the efficacy of a combined approach involving picosecond-resolved FRET and polarisation-gated fluorescence for the exploration of ultrafast dynamics in the allosteric interaction of the Gal repressor (GalR) protein dimer with DNA operator sequences OE and OI . FRET from the single tryptophan residue to a covalently attached probe IAEDANS at a cysteine residue in the C-terminal domain of GalR shows structural perturbation and conformational dynamics during allosteric interaction. Polarisation-gated fluorescence spectroscopy of IAEDANS and another probe (FITC) covalently attached to the operator directly revealed the essential dynamics for cooperativity in the protein-protein interaction. The ultrafast resonance energy transfer from IAEDANS in the protein to FITC also revealed different dynamic flexibility in the allosteric interaction. An attempt was made to correlate the dynamic changes in the protein dimers with OE and OI with the consequent protein-protein interaction (tetramerisation) to form a DNA loop encompassing the promoter segment.

  11. Dioxin Exposure Blocks Lactation through a Direct Effect on Mammary Epithelial Cells Mediated by the Aryl Hydrocarbon Receptor Repressor

    PubMed Central

    Basham, Kaitlin J.; Leonard, Christopher J.; Kieffer, Collin; Shelton, Dawne N.; McDowell, Maria E.; Bhonde, Vasudev R.; Looper, Ryan E.; Welm, Bryan E.

    2015-01-01

    In mammals, lactation is a rich source of nutrients and antibodies for newborn animals. However, millions of mothers each year experience an inability to breastfeed. Exposure to several environmental toxicants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been strongly implicated in impaired mammary differentiation and lactation. TCDD and related polyhalogenated aromatic hydrocarbons are widespread industrial pollutants that activate the aryl hydrocarbon receptor (AHR). Despite many epidemiological and animal studies, the molecular mechanism through which AHR signaling blocks lactation remains unclear. We employed in vitro models of mammary differentiation to recapitulate lactogenesis in the presence of toxicants. We demonstrate AHR agonists directly block milk production in isolated mammary epithelial cells. Moreover, we define a novel role for the aryl hydrocarbon receptor repressor (AHRR) in mediating this response. Our mechanistic studies suggest AHRR is sufficient to block transcription of the milk gene β-casein. As TCDD is a prevalent environmental pollutant that affects women worldwide, our results have important public health implications for newborn nutrition. PMID:25265996

  12. Molecular mechanism of quinone signaling mediated through S-quinonization of a YodB family repressor QsrR

    PubMed Central

    Ji, Quanjiang; Zhang, Liang; Jones, Marcus B.; Sun, Fei; Deng, Xin; Liang, Haihua; Cho, Hoonsik; Brugarolas, Pedro; Gao, Yihe N.; Peterson, Scott N.; Lan, Lefu; Bae, Taeok; He, Chuan

    2013-01-01

    Quinone molecules are intracellular electron-transport carriers, as well as critical intra- and extracellular signals. However, transcriptional regulation of quinone signaling and its molecular basis are poorly understood. Here, we identify a thiol-stress-sensing regulator YodB family transcriptional regulator as a central component of quinone stress response of Staphylococcus aureus, which we have termed the quinone-sensing and response repressor (QsrR). We also identify and confirm an unprecedented quinone-sensing mechanism based on the S-quinonization of the essential residue Cys-5. Structural characterizations of the QsrR–DNA and QsrR–menadione complexes further reveal that the covalent association of menadione directly leads to the release of QsrR from operator DNA following a 10° rigid-body rotation as well as a 9-Å elongation between the dimeric subunits. The molecular level characterization of this quinone-sensing transcriptional regulator provides critical insights into quinone-mediated gene regulation in human pathogens. PMID:23479646

  13. T Cell Fates Zipped Up: How the Bach2 Basic Leucine Zipper Transcriptional Repressor Directs T Cell Differentiation and Function.

    PubMed

    Richer, Martin J; Lang, Mark L; Butler, Noah S

    2016-08-15

    Recent data illustrate a key role for the transcriptional regulator bric-a-brac, tramtrack, and broad complex and cap'n'collar homology (Bach)2 in orchestrating T cell differentiation and function. Although Bach2 has a well-described role in B cell differentiation, emerging data show that Bach2 is a prototypical member of a novel class of transcription factors that regulates transcriptional activity in T cells at super-enhancers, or regions of high transcriptional activity. Accumulating data demonstrate specific roles for Bach2 in favoring regulatory T cell generation, restraining effector T cell differentiation, and potentiating memory T cell development. Evidence suggests that Bach2 regulates various facets of T cell function by repressing other key transcriptional regulators such as B lymphocyte-induced maturation protein 1. In this review, we examine our present understanding of the role of Bach2 in T cell function and highlight the growing evidence that this transcriptional repressor functions as a key regulator involved in maintenance of T cell quiescence, T cell subset differentiation, and memory T cell generation. PMID:27496973

  14. Phosphorylation of the chromatin remodeling factor DPF3a induces cardiac hypertrophy through releasing HEY repressors from DNA

    PubMed Central

    Cui, Huanhuan; Schlesinger, Jenny; Schoenhals, Sophia; Tönjes, Martje; Dunkel, Ilona; Meierhofer, David; Cano, Elena; Schulz, Kerstin; Berger, Michael F.; Haack, Timm; Abdelilah-Seyfried, Salim; Bulyk, Martha L.; Sauer, Sascha; Sperling, Silke R.

    2016-01-01

    DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure. PMID:26582913

  15. The Hinge Region Strengthens the Nonspecific Interaction between Lac-Repressor and DNA: A Computer Simulation Study

    PubMed Central

    Sun, Lili; Tabaka, Marcin; Hou, Sen; Li, Lin; Burdzy, Krzysztof; Aksimentiev, Aleksei; Maffeo, Christopher; Zhang, Xuzhu; Holyst, Robert

    2016-01-01

    LacI is commonly used as a model to study the protein-DNA interaction and gene regulation. The headpiece of the lac-repressor (LacI) protein is an ideal system for investigation of nonspecific binding of the whole LacI protein to DNA. The hinge region of the headpiece has been known to play a key role in the specific binding of LacI to DNA, whereas its role in nonspecific binding process has not been elucidated. Here, we report the results of explicit solvent molecular dynamics simulation and continuum electrostatic calculations suggesting that the hinge region strengthens the nonspecific interaction, accounting for up to 50% of the micro-dissociation free energy of LacI from DNA. Consequently, the rate of microscopic dissociation of LacI from DNA is reduced by 2~3 orders of magnitude in the absence of the hinge region. We find the hinge region makes an important contribution to the electrostatic energy, the salt dependence of electrostatic energy, and the number of salt ions excluded from binding of the LacI-DNA complex. PMID:27008630

  16. Radiation response in Deinococcus deserti: IrrE is a metalloprotease that cleaves repressor protein DdrO.