Differential transcriptional regulation by alternatively designed mechanisms: A mathematical modeling approach.

PubMed

Yildirim, Necmettin; Aktas, Mehmet Emin; Ozcan, Seyma Nur; Akbas, Esra; Ay, Ahmet

2017-01-01

Cells maintain cellular homeostasis employing different regulatory mechanisms to respond external stimuli. We study two groups of signal-dependent transcriptional regulatory mechanisms. In the first group, we assume that repressor and activator proteins compete for binding to the same regulatory site on DNA (competitive mechanisms). In the second group, they can bind to different regulatory regions in a noncompetitive fashion (noncompetitive mechanisms). For both competitive and noncompetitive mechanisms, we studied the gene expression dynamics by increasing the repressor or decreasing the activator abundance (inhibition mechanisms), or by decreasing the repressor or increasing the activator abundance (activation mechanisms). We employed delay differential equation models. Our simulation results show that the competitive and noncompetitive inhibition mechanisms exhibit comparable repression effectiveness. However, response time is fastest in the noncompetitive inhibition mechanism due to increased repressor abundance, and slowest in the competitive inhibition mechanism by increased repressor level. The competitive and noncompetitive inhibition mechanisms through decreased activator abundance show comparable and moderate response times, while the competitive and noncompetitive activation mechanisms by increased activator protein level display more effective and faster response. Our study exemplifies the importance of mathematical modeling and computer simulation in the analysis of gene expression dynamics.

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

PubMed Central

Zhao, Xiao; Hume, Samantha L; Johnson, Christopher; Thompson, Paul; Huang, Junyong; Gray, Joe; Lamb, Heather K; Hawkins, Alastair R

2010-01-01

The role of specific cleavage of transcription repressor proteins by proteases and how this may be related to the emerging theme of dinucleotides as cellular signaling molecules is poorly characterized. The transcription repressor NmrA of Aspergillus nidulans discriminates between oxidized and reduced dinucleotides, however, dinucleotide binding has no effect on its interaction with the zinc finger in the transcription activator AreA. Protease activity in A. nidulans was assayed using NmrA as the substrate, and was absent in mycelium grown under nitrogen sufficient conditions but abundant in mycelium starved of nitrogen. One of the proteases was purified and identified as the protein Q5BAR4 encoded by the gene AN2366.2. Fluorescence confocal microscopy showed that the nuclear levels of NmrA were reduced approximately 38% when mycelium was grown on nitrate compared to ammonium and absent when starved of nitrogen. Proteolysis of NmrA occurred in an ordered manner by preferential digestion within a C-terminal surface exposed loop and subsequent digestion at other sites. NmrA digested at the C-terminal site was unable to bind to the AreA zinc finger. These data reveal a potential new layer of control of nitrogen metabolite repression by the ordered proteolytic cleavage of NmrA. NmrA digested at the C-terminal site retained the ability to bind NAD+ and showed a resistance to further digestion that was enhanced by the presence of NAD+. This is the first time that an effect of dinucleotide binding to NmrA has been demonstrated. PMID:20506376

Structural and functional analysis of the repressor complex in the Notch signaling pathway of Drosophila melanogaster

PubMed Central

Maier, Dieter; Kurth, Patricia; Schulz, Adriana; Russell, Andrew; Yuan, Zhenyu; Gruber, Kim; Kovall, Rhett A.; Preiss, Anette

2011-01-01

In metazoans, the highly conserved Notch pathway drives cellular specification. On receptor activation, the intracellular domain of Notch assembles a transcriptional activator complex that includes the DNA-binding protein CSL, a composite of human C-promoter binding factor 1, Suppressor of Hairless of Drosophila melanogaster [Su(H)], and lin-12 and Glp-1 phenotype of Caenorhabditis elegans. In the absence of ligand, CSL represses Notch target genes. However, despite the structural similarity of CSL orthologues, repression appears largely diverse between organisms. Here we analyze the Notch repressor complex in Drosophila, consisting of the fly CSL protein, Su(H), and the corepressor Hairless, which recruits general repressor proteins. We show that the C-terminal domain of Su(H) is necessary and sufficient for forming a high-affinity complex with Hairless. Mutations in Su(H) that affect interactions with Notch and Mastermind have no effect on Hairless binding. Nonetheless, we demonstrate that Notch and Hairless compete for CSL in vitro and in cell culture. In addition, we identify a site in Hairless that is crucial for binding Su(H) and subsequently show that this Hairless mutant is strongly impaired, failing to properly assemble the repressor complex in vivo. Finally, we demonstrate Hairless-mediated inhibition of Notch signaling in a cell culture assay, which hints at a potentially similar repression mechanism in mammals that might be exploited for therapeutic purposes. PMID:21737682

Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor l-arginine

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

Garnett, James A.; Baumberg, Simon; Stockley, Peter G.

2007-11-01

The crystal structure of the C-terminal domain hexameric core of AhrC, with bound corepressor (l-arginine), has been solved at 1.95 Å resolution. Binding of l-arginine results in a rotation between the two trimers of the hexamer, leading to the activation of the DNA-binding state. The arginine repressor/activator protein (AhrC) from Bacillus subtilis belongs to a large family of multifunctional transcription factors that are involved in the regulation of bacterial arginine metabolism. AhrC interacts with operator sites in the promoters of arginine biosynthetic and catabolic operons, acting as a transcriptional repressor at biosynthetic sites and an activator of transcription at catabolicmore » sites. AhrC is a hexamer of identical subunits, each having two domains. The C-terminal domains form the core of the protein and are involved in oligomerization and l-arginine binding. The N-terminal domains lie on the outside of the compact core and play a role in binding to 18 bp DNA operators called ARG boxes. The C-terminal domain of AhrC has been expressed, purified and characterized, and also crystallized as a hexamer with the bound corepressor l-arginine. Here, the crystal structure refined to 1.95 Å is presented.« less

Expression profiling of clonal lymphocyte cell cultures from Rett syndrome patients

USDA-ARS?s Scientific Manuscript database

More than 85% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked MECP2 gene which encodes methyl-CpG-binding protein 2, a transcriptional repressor that binds methylated CpG sites. Because MECP2 is subject to X chromosome inactivation (XCI), girls with RTT express either the...

Structural Explanation for Allolactose (lac Operon Inducer) Synthesis by lacZ β-Galactosidase and the Evolutionary Relationship between Allolactose Synthesis and the lac Repressor

PubMed Central

Wheatley, Robert W.; Lo, Summie; Jancewicz, Larisa J.; Dugdale, Megan L.; Huber, Reuben E.

2013-01-01

β-Galactosidase (lacZ) has bifunctional activity. It hydrolyzes lactose to galactose and glucose and catalyzes the intramolecular isomerization of lactose to allolactose, the lac operon inducer. β-Galactosidase promotes the isomerization by means of an acceptor site that binds glucose after its cleavage from lactose and thus delays its exit from the site. However, because of its relatively low affinity for glucose, details of this site have remained elusive. We present structural data mapping the glucose site based on a substituted enzyme (G794A-β-galactosidase) that traps allolactose. Various lines of evidence indicate that the glucose of the trapped allolactose is in the acceptor position. The evidence includes structures with Bis-Tris (2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol) and l-ribose in the site and kinetic binding studies with substituted β-galactosidases. The site is composed of Asn-102, His-418, Lys-517, Ser-796, Glu-797, and Trp-999. Ser-796 and Glu-797 are part of a loop (residues 795–803) that closes over the active site. This loop appears essential for the bifunctional nature of the enzyme because it helps form the glucose binding site. In addition, because the loop is mobile, glucose binding is transient, allowing the release of some glucose. Bioinformatics studies showed that the residues important for interacting with glucose are only conserved in a subset of related enzymes. Thus, intramolecular isomerization is not a universal feature of β-galactosidases. Genomic analyses indicated that lac repressors were co-selected only within the conserved subset. This shows that the glucose binding site of β-galactosidase played an important role in lac operon evolution. PMID:23486479

Interplay of protein and DNA structure revealed in simulations of the lac operon.

PubMed

Czapla, Luke; Grosner, Michael A; Swigon, David; Olson, Wilma K

2013-01-01

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

Regulation of neural macroRNAs by the transcriptional repressor REST

PubMed Central

Johnson, Rory; Teh, Christina Hui-Leng; Jia, Hui; Vanisri, Ravi Raj; Pandey, Tridansh; Lu, Zhong-Hao; Buckley, Noel J.; Stanton, Lawrence W.; Lipovich, Leonard

2009-01-01

The essential transcriptional repressor REST (repressor element 1-silencing transcription factor) plays central roles in development and human disease by regulating a large cohort of neural genes. These have conventionally fallen into the class of known, protein-coding genes; recently, however, several noncoding microRNA genes were identified as REST targets. Given the widespread transcription of messenger RNA-like, noncoding RNAs (“macroRNAs”), some of which are functional and implicated in disease in mammalian genomes, we sought to determine whether this class of noncoding RNAs can also be regulated by REST. By applying a new, unbiased target gene annotation pipeline to computationally discovered REST binding sites, we find that 23% of mammalian REST genomic binding sites are within 10 kb of a macroRNA gene. These putative target genes were overlooked by previous studies. Focusing on a set of 18 candidate macroRNA targets from mouse, we experimentally demonstrate that two are regulated by REST in neural stem cells. Flanking protein-coding genes are, at most, weakly repressed, suggesting specific targeting of the macroRNAs by REST. Similar to the majority of known REST target genes, both of these macroRNAs are induced during nervous system development and have neurally restricted expression profiles in adult mouse. We observe a similar phenomenon in human: the DiGeorge syndrome-associated noncoding RNA, DGCR5, is repressed by REST through a proximal upstream binding site. Therefore neural macroRNAs represent an additional component of the REST regulatory network. These macroRNAs are new candidates for understanding the role of REST in neuronal development, neurodegeneration, and cancer. PMID:19050060

Regulation of neural macroRNAs by the transcriptional repressor REST.

PubMed

Johnson, Rory; Teh, Christina Hui-Leng; Jia, Hui; Vanisri, Ravi Raj; Pandey, Tridansh; Lu, Zhong-Hao; Buckley, Noel J; Stanton, Lawrence W; Lipovich, Leonard

2009-01-01

The essential transcriptional repressor REST (repressor element 1-silencing transcription factor) plays central roles in development and human disease by regulating a large cohort of neural genes. These have conventionally fallen into the class of known, protein-coding genes; recently, however, several noncoding microRNA genes were identified as REST targets. Given the widespread transcription of messenger RNA-like, noncoding RNAs ("macroRNAs"), some of which are functional and implicated in disease in mammalian genomes, we sought to determine whether this class of noncoding RNAs can also be regulated by REST. By applying a new, unbiased target gene annotation pipeline to computationally discovered REST binding sites, we find that 23% of mammalian REST genomic binding sites are within 10 kb of a macroRNA gene. These putative target genes were overlooked by previous studies. Focusing on a set of 18 candidate macroRNA targets from mouse, we experimentally demonstrate that two are regulated by REST in neural stem cells. Flanking protein-coding genes are, at most, weakly repressed, suggesting specific targeting of the macroRNAs by REST. Similar to the majority of known REST target genes, both of these macroRNAs are induced during nervous system development and have neurally restricted expression profiles in adult mouse. We observe a similar phenomenon in human: the DiGeorge syndrome-associated noncoding RNA, DGCR5, is repressed by REST through a proximal upstream binding site. Therefore neural macroRNAs represent an additional component of the REST regulatory network. These macroRNAs are new candidates for understanding the role of REST in neuronal development, neurodegeneration, and cancer.

PdhR, the pyruvate dehydrogenase repressor, does not regulate lipoic acid synthesis.

PubMed

Feng, Youjun; Cronan, John E

2014-01-01

Lipoic acid is a covalently-bound enzyme cofactor required for central metabolism all three domains of life. In the last 20 years the pathway of lipoic acid synthesis and metabolism has been established in Escherichia coli. Expression of the genes of the lipoic acid biosynthesis pathway was believed to be constitutive. However, in 2010 Kaleta and coworkers (BMC Syst. Biol. 4:116) predicted a binding site for the pyruvate dehydrogenase operon repressor, PdhR (referred to lipA site 1) upstream of lipA, the gene encoding lipoic acid synthase and concluded that PdhR regulates lipA transcription. We report in vivo and in vitro evidence that lipA is not controlled by PdhR and that the putative regulatory site deduced by the prior workers is nonfunctional and physiologically irrelevant. E. coli PdhR was purified to homogeneity and used for electrophoretic mobility shift assays. The lipA site 1 of Kaleta and coworkers failed to bind PdhR. The binding detected by these workers is due to another site (lipA site 3) located far upstream of the lipA promoter. Relative to the canonical PdhR binding site lipA site 3 is a half-palindrome and as expected had only weak PdhR binding ability. Manipulation of lipA site 3 to construct a palindrome gave significantly enhanced PdhR binding affinity. The native lipA promoter and the version carrying the artificial lipA3 palindrome were transcriptionally fused to a LacZ reporter gene to directly assay lipA expression. Deletion of pdhR gave no significant change in lipA promoter-driven β-galactosidase activity with either the native or constructed palindrome upstream sequences, indicating that PdhR plays no physiological role in regulation of lipA expression. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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

PubMed

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

2012-01-01

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

Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors

PubMed Central

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

2012-01-01

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression. PMID:22615585

Stochastic model of template-directed elongation processes in biology.

PubMed

Schilstra, Maria J; Nehaniv, Chrystopher L

2010-10-01

We present a novel modular, stochastic model for biological template-based linear chain elongation processes. In this model, elongation complexes (ECs; DNA polymerase, RNA polymerase, or ribosomes associated with nascent chains) that span a finite number of template units step along the template, one after another, with semaphore constructs preventing overtaking. The central elongation module is readily extended with modules that represent initiation and termination processes. The model was used to explore the effect of EC span on motor velocity and dispersion, and the effect of initiation activator and repressor binding kinetics on the overall elongation dynamics. The results demonstrate that (1) motors that move smoothly are able to travel at a greater velocity and closer together than motors that move more erratically, and (2) the rate at which completed chains are released is proportional to the occupancy or vacancy of activator or repressor binding sites only when initiation or activator/repressor dissociation is slow in comparison with elongation. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Interference between Triplex and Protein Binding to Distal Sites on Supercoiled DNA.

PubMed

Noy, Agnes; Maxwell, Anthony; Harris, Sarah A

2017-02-07

We have explored the interdependence of the binding of a DNA triplex and a repressor protein to distal recognition sites on supercoiled DNA minicircles using MD simulations. We observe that the interaction between the two ligands through their influence on their DNA template is determined by a subtle interplay of DNA mechanics and electrostatics, that the changes in flexibility induced by ligand binding play an important role and that supercoiling can instigate additional ligand-DNA contacts that would not be possible in simple linear DNA sequences. Copyright © 2017. Published by Elsevier Inc.

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

Nishino, Tomonori G.; Department of Biotechnology, University of Tokyo, Bunkyo-ku, Tokyo 113-8657; Miyazaki, Masaya

Class IIa histone deacetylases (HDACs) form complexes with a class of transcriptional repressors in the nucleus. While screening for compounds that could block the association of HDAC4 with the BTB domain-containing transcriptional repressor Bach2, we discovered that phorbol 12-myristate 13-acetate (PMA) induced the cytoplasmic retention of HDAC4 mutants lacking a nuclear export signal (NES). Although PMA treatment and PKD overexpression has been proposed to facilitate the nuclear export of class IIa HDACs by creating 14-3-3 binding sites containing phosphoserines, our experiments using HDAC mutants demonstrated that PMA greatly reduces nuclear import. PMA treatment repressed the NLS activity in a mannermore » dependent on 14-3-3 binding. These results suggest that nuclear HDAC4 is not tethered in the nucleus, but instead shuttles between the nucleus and the cytoplasm. Phosphorylation-induced 14-3-3 binding biases the balance of nucleo-cytoplasmic shuttling toward the cytoplasm by inhibiting nuclear import.« less

Information analysis of sequences that bind the replication initiator RepA | Center for Cancer Research

Cancer.gov

The tall letters represent the highly conserved bases in DNA binding sites of several prokaryotic repressors and activators. Conservation is strongest where major grooves of the double helical DNA (represented by crests of a cosine wave) face the protein. This shows that conservation analysis alone can be used to predict the face of DNA that contacts the proteins.

A calorimetric investigation of the interaction of the lac repressor with inducer.

PubMed

Donnér, J; Caruthers, M H; Gill, S J

1982-12-25

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.

A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

PubMed

Håvik, B; Ragnhildstveit, E; Lorens, J B; Saelemyr, K; Fauske, O; Knudsen, L K; Fjose, A

1999-12-20

The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences. Copyright 1999 Academic Press.

Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding.

PubMed

Subrahmanyam, S; Cronan, J E

1999-01-21

We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein. This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis. By the use of a radioactively labeled DNA-binding protein and nonradioactive DNA coupled with PCR amplification from gel slices, we show that specific binding sites can be isolated from Escherichia coli genomic DNA. We have applied this method to isolate a binding site for FadR, a global regulator of fatty acid metabolism in E. coli. We have also isolated a second binding site for BirA, the biotin operon repressor/biotin ligase, from the E. coli genome that has a very low binding efficiency compared with the bio operator region.

Structure of the MecI repressor from Staphylococcus aureus in complex with the cognate DNA operator of mec

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

Safo, Martin K., E-mail: msafo@vcu.edu; Ko, Tzu-Ping; Musayev, Faik N.

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,more » 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.« less

Solution structure of Mycobacterium tuberculosis NmtR in the apo-state: Insights into Ni(II)-mediated allostery

PubMed Central

Lee, Chul Won; Chakravorty, Dhruva K.; Chang, Feng-Ming James; Reyes-Caballero, Hermes; Ye, Yuzhen; Merz, Kenneth M.; Giedroc, David P.

2012-01-01

Mycobacterium tuberculosis is an obligate human respiratory pathogen that encodes approximately ten arsenic repressor (ArsR) family regulatory proteins that allow the organism to respond to a wide range of changes in its immediate microenvironment. How individual ArsR repressors have evolved to respond to selective stimuli is of intrinsic interest. The Ni(II)/Co(II)-specific repressor NmtR and related actinomycete nickel sensors harbor a conserved N-terminal αNH2-Gly2-His3-Gly4 sequence. Here, we present the solution structure of homodimeric apo-NmtR and show that the core of the molecule adopts a typical winged-helix ArsR repressor (α1-α2-α3-αR-β1-β2-α5) “open conformation” that is similar to the related zinc sensor Staphylococcus aureus CzrA, but harboring long, flexible N-terminal (residues 2-16) and C-terminal (residues 109-120) extensions. Ni(II) binding to the regulatory sites induces strong paramagnetic broadening of the α5 helical region and the extreme N-terminal tail to residue 10. Ratiometric pulse chase amidination mass spectrometry reveals that the rate of amidination of the Gly2 α-amino group is strongly attenuated in the Ni(II) complex relative to the apo-state and non-cognate Zn(II) complex. Ni(II) binding also induces dynamic disorder in the μs-ms timescale of key DNA interacting regions that likely contributes to the negative regulation of DNA binding by Ni(II). Molecular dynamics simulations and quantum chemical calculations reveal that NmtR readily accommodates a distal Ni(II) hexacoordination model involving the α-amine and His3 of the N-terminal region and α5 residues Asp91′, His93′, His104 and His107, which collectively define a new metal sensing site configuration in ArsR family regulators. PMID:22394357

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

PubMed Central

Jennings, Barbara H.

2014-01-01

Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase). We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in “active” chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation and polymerase pausing. PMID:25165826

DNA wrapping and distortion by an oligomeric homeodomain protein.

PubMed

Williams, Hannah; Jayaraman, Padma-Sheela; Gaston, Kevin

2008-10-31

Many transcription factors alter DNA or chromatin structure. Changes in chromatin structure are often brought about by the recruitment of chromatin-binding proteins, chromatin-modifying proteins, or other transcription co-activator or co-repressor proteins. However, some transcription factors form oligomeric assemblies that may themselves induce changes in DNA conformation and chromatin structure. The proline-rich homeodomain (PRH/Hex) protein is a transcription factor that regulates cell differentiation and cell proliferation, and has multiple roles in embryonic development. Earlier, we showed that PRH can repress transcription by multiple mechanisms, including the recruitment of co-repressor proteins belonging to the TLE family of chromatin-binding proteins. Our in vivo crosslinking studies have shown that PRH forms oligomeric complexes in cells and a variety of biophysical techniques suggest that the protein forms octamers. However, as yet we have little knowledge of the role played by PRH oligomerisation in the regulation of promoter activity or of the architecture of promoters that are regulated directly by PRH in cells. Here, we compare the binding of PRH and the isolated PRH homeodomain to DNA fragments with single and multiple PRH sites, using gel retardation assays and DNase I and chemical footprinting. We show that the PRH oligomer binds to multiple sites within the human Goosecoid promoter with high affinity and that the binding of PRH brings about DNA distortion. We suggest that PRH octamers wrap DNA in order to bring about transcriptional repression.

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. Copyright © 2014. Published by Elsevier B.V.

TGF-{beta} signals the formation of a unique NF1/Smad4-dependent transcription repressor-complex in human diploid fibroblasts

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

Luciakova, Katarina, E-mail: katarina.luciakova@savba.sk; Kollarovic, Gabriel; Kretova, Miroslava

2011-08-05

Highlights: {yields} TGF-{beta} induces the formation of unique nuclear NF1/Smad4 complexes that repress expression of the ANT-2 gene. {yields} Repression is mediated through an NF1-dependent repressor element in the promoter. {yields} The formation of NF1/Smad4 complexes and the repression of ANT2 are prevented by inhibitors of p38 kinase and TGF-{beta} RI. {yields} NF1/Smad complexes implicate novel role for NF1 and Smad proteins in the regulation of growth. -- Abstract: We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G.more » Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123-130]. In the present study, we show that TGF-{beta}, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-{beta} is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-{beta} are prevented by inhibitors of TGF-{beta} RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-{beta} and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-{beta} signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state.« less

A high-resolution structure of the DNA-binding domain of AhrC, the arginine repressor/activator protein from Bacillus subtilis

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

Garnett, James A.; Baumberg, Simon; Stockley, Peter G.

2007-11-01

The structure of the winged helix–turn–helix DNA-binding domain of AhrC has been determined at 1.0 Å resolution. The largely hydrophobic β-wing shows high B factors and may mediate the dimer interface in operator complexes. In Bacillus subtilis the concentration of l-arginine is controlled by the transcriptional regulator AhrC, which interacts with 18 bp DNA operator sites called ARG boxes in the promoters of arginine biosynthetic and catabolic operons. AhrC is a 100 kDa homohexamer, with each subunit having two domains. The C-terminal domains form the core, mediating intersubunit interactions and binding of the co-repressor l-arginine, whilst the N-terminal domains containmore » a winged helix–turn–helix DNA-binding motif and are arranged around the periphery. The N-terminal domain of AhrC has been expressed, purified and characterized and it has been shown that the fragment still binds DNA operators as a recombinant monomer. The DNA-binding domain has also been crystallized and the crystal structure refined to 1.0 Å resolution is presented.« less

The global repressor FliZ antagonizes gene expression by σS-containing RNA polymerase due to overlapping DNA binding specificity.

PubMed

Pesavento, Christina; Hengge, Regine

2012-06-01

FliZ, a global regulatory protein under the control of the flagellar master regulator FlhDC, was shown to antagonize σ(S)-dependent gene expression in Escherichia coli. Thereby it plays a pivotal role in the decision between alternative life-styles, i.e. FlhDC-controlled flagellum-based motility or σ(S)-dependent curli fimbriae-mediated adhesion and biofilm formation. Here, we show that FliZ is an abundant DNA-binding protein that inhibits gene expression mediated by σ(S) by recognizing operator sequences that resemble the -10 region of σ(S)-dependent promoters. FliZ does so with a structural element that is similar to region 3.0 of σ(S). Within this element, R108 in FliZ corresponds to K173 in σ(S), which contacts a conserved cytosine at the -13 promoter position that is specific for σ(S)-dependent promoters. R108 as well as C(-13) are also crucial for DNA binding by FliZ. However, while a number of FliZ binding sites correspond to known σ(S)-dependent promoters, promoter activity is not a prerequisite for FliZ binding and repressor function. Thus, we demonstrate that FliZ also feedback-controls flagellar gene expression by binding to a site in the flhDC control region that shows similarity only to a -10 element of a σ(S)-dependent promoter, but does not function as a promoter.

The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells

PubMed Central

2010-01-01

Background The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function. Results A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636 consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1-ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells. Conclusions We demonstrate that the GATA-1 transcription factor binds and transactivates the ETO proximal promoter in an erythroid/megakaryocytic-specific manner. Thus, trans-acting factors that are essential in erythroid/megakaryocytic differentiation govern ETO expression. PMID:20487545

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

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

Goffinont, S.; Davidkova, M.; Spotheim-Maurizot, M., E-mail: spotheim@cnrs-orleans.fr

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 leucinemore » residues of the tetramerization domain.« less

Differential regulation of transcription through distinct Suppressor of Hairless DNA binding site architectures during Notch signaling in proneural clusters.

PubMed

Cave, John W; Xia, Li; Caudy, Michael

2011-01-01

In Drosophila melanogaster, achaete (ac) and m8 are model basic helix-loop-helix activator (bHLH A) and repressor genes, respectively, that have the opposite cell expression pattern in proneural clusters during Notch signaling. Previous studies have shown that activation of m8 transcription in specific cells within proneural clusters by Notch signaling is programmed by a "combinatorial" and "architectural" DNA transcription code containing binding sites for the Su(H) and proneural bHLH A proteins. Here we show the novel result that the ac promoter contains a similar combinatorial code of Su(H) and bHLH A binding sites but contains a different Su(H) site architectural code that does not mediate activation during Notch signaling, thus programming a cell expression pattern opposite that of m8 in proneural clusters.

AmrZ Beta-Sheet Residues Are Essential for DNA Binding and Transcriptional Control of Pseudomonas aeruginosa Virulence Genes ▿ †

PubMed Central

Waligora, Elizabeth A.; Ramsey, Deborah M.; Pryor, Edward E.; Lu, Haiping; Hollis, Thomas; Sloan, Gina P.; Deora, Rajendar; Wozniak, Daniel J.

2010-01-01

AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the β-sheet for DNA binding, while the α-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a β-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ β-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection. PMID:20709902

Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

PubMed

Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J

2000-12-01

The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

Salmonella typhimurium PtsJ is a novel MocR-like transcriptional repressor involved in regulating the vitamin B6 salvage pathway.

PubMed

Tramonti, Angela; Milano, Teresa; Nardella, Caterina; di Salvo, Martino L; Pascarella, Stefano; Contestabile, Roberto

2017-02-01

The vitamin B 6 salvage pathway, involving pyridoxine 5'-phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B 6 vitamers from nutrients and protein turnover to produce pyridoxal 5'-phosphate (PLP), the catalytically active form of the vitamin. Regulation of this pathway, widespread in living organisms including humans and many bacteria, is very important to vitamin B 6 homeostasis but poorly understood. Although some information is available on the enzymatic regulation of PNPOx and PLK, little is known on their regulation at the transcriptional level. In the present work, we identified a new MocR-like regulator, PtsJ from Salmonella typhimurium, which controls the expression of the pdxK gene encoding one of the two PLKs expressed in this organism (PLK1). Analysis of pdxK expression in a ptsJ knockout strain demonstrated that PtsJ acts as a transcriptional repressor. This is the first case of a MocR-like regulator acting as repressor of its target gene. Expression and purification of PtsJ allowed a detailed characterisation of its effector and DNA-binding properties. PLP is the only B 6 vitamer acting as effector molecule for PtsJ. A DNA-binding region composed of four repeated nucleotide sequences is responsible for binding of PtsJ to its target promoter. Analysis of binding stoichiometry revealed that protein subunits/DNA molar ratio varies from 4 : 1 to 2 : 1, depending on the presence or absence of PLP. Structural characteristics of DNA transcriptional factor-binding sites suggest that PtsJ binds DNA according to a different model with respect to other characterised members of the MocR subgroup. © 2016 Federation of European Biochemical Societies.

Stress-Responsive Mitogen-Activated Protein Kinases Interact with the EAR Motif of a Poplar Zinc Finger Protein and Mediate Its Degradation through the 26S Proteasome1[W][OA

PubMed Central

Hamel, Louis-Philippe; Benchabane, Meriem; Nicole, Marie-Claude; Major, Ian T.; Morency, Marie-Josée; Pelletier, Gervais; Beaudoin, Nathalie; Sheen, Jen; Séguin, Armand

2011-01-01

Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors. PMID:21873571

In vivo estradiol-dependent dephosphorylation of the repressor MDBP-2-H1 correlates with the loss of in vitro preferential binding to methylated DNA.

PubMed Central

Bruhat, A; Jost, J P

1995-01-01

We have previously shown that estradiol treatment of roosters resulted in a rapid loss of binding activity of the repressor MDBP-2-H1 (a member of the histone H1 family) to methylated DNA that was not due to a decrease in MDBP-2-H1 concentration. Here we demonstrate that MDBP-2-H1 from rooster liver nuclear extracts is a phosphoprotein. Phosphoamino acid analysis reveals that the phosphorylation occurs exclusively on serine residues. Two-dimensional gel electrophoresis and tryptic phosphopeptide analysis show that MDBP-2-H1 is phosphorylated at several sites. Treatment of roosters with estradiol triggers a dephosphorylation of at least two sites in the protein. Phosphatase treatment of purified rooster MDBP-2-H1 combined with gel mobility shift assay indicates that phosphorylation of MDBP-2-H1 is essential for the binding to methylated DNA and that the dephosphorylation can occur on the protein bound to methylated DNA causing its release from DNA. Thus, these results suggest that in vivo modification of the phosphorylation status of MDBP-2-H1 caused by estradiol treatment may be a key step for the down regulation of its binding to methylated DNA. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7731964

Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate promoters.

PubMed

Feng, Youjun; Cronan, John E

2011-04-01

Two transcriptional regulators, the FadR activator and the FabR repressor, control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was blocked in the absence of exogenous unsaturated fatty acids in a ΔfadR strain and found that the rates of transcription of fabA and fabB were unaffected by the lack of unsaturated thioesters. To examine the discrepancy between our in vivo results and the prior in vitro results we obtained active, natively folded forms of the E. coli and Vibrio cholerae FabRs by use of an in vitro transcription-translation system. We report that FabR bound the intact promoter regions of both fabA and fabB in the absence of unsaturated acyl thioesters, but bound the two promoters differently. Native FabR bound the fabA promoter region provided that the canonical FabR binding site is extended by inclusion of flanking sequences that overlap the neighbouring FadR binding site. In contrast, although binding to the fabB operator also required a flanking sequence, a non-specific sequence could suffice. However, unsaturated thioesters did allow FabR binding to the minimal FabR operator sites of both promoters which otherwise were not bound. Thus unsaturated thioester ligands were not essential for FabR/target DNA interaction, but acted to enhance binding. The gel mobility shift data plus in vivo expression data indicate that despite the remarkably similar arrangements of promoter elements, FadR predominately regulates fabA expression whereas FabR is the dominant regulator of fabB expression. We also report that E. coli fabR expression is not autoregulated. Complementation, qRT-PCR and fatty acid composition analyses demonstrated that V. cholerae FabR was a functional repressor of unsaturated fatty acid synthesis. However, in contrast to E. coli, gel mobility shift assays indicated that neither E. coli nor V. cholerae FabRs bound the V. cholerae fabB promoter, although both proteins efficiently bound the V. cholerae fabA promoter. This asymmetry was shown to be due to the lack of a FabR binding site within the V. cholerae fabB promoter region. © 2011 Blackwell Publishing Ltd.

Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

PubMed

Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J; Quinlan, Kate G R; Cordwell, Stuart J; Funnell, Alister P W; Pearson, Richard C M; Crossley, Merlin

2015-03-27

Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure of the Mecl Repressor from Staphylococcus aureus in Complex with the Cognate DNA Operator of mec

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

Safo,M.; Ko, T.; Musayev, F.

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 virtualmore » 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.« less

Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

PubMed

Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

2014-03-14

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

PubMed

Goldman, D; Sapru, M K; Stewart, S; Plotkin, J; Libermann, T A; Wasylyk, B; Guan, K

1998-10-15

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

Specific repression of β-globin promoter activity by nuclear ferritin

PubMed Central

Broyles, Robert H.; Belegu, Visar; DeWitt, Christina R.; Shah, Sandeep N.; Stewart, Charles A.; Pye, Quentin N.; Floyd, Robert A.

2001-01-01

Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult β-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the β-globin promoter at −153 to −148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress β-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the β-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant β-globin promoter for the −150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the −150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human β-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases. PMID:11481480

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

PubMed Central

Busby, Jason N.; Fritz, Georg; Moreland, Nicole J.; Cook, Gregory M.; Lott, J. Shaun; Baker, Edward N.

2014-01-01

Bacterial uptake of phosphate is usually accomplished via high-affinity transporters that are commonly regulated by two-component systems, which are activated when the concentration of phosphate is low. Mycobacterium smegmatis possesses two such transporters, the widely distributed PstSCAB system and PhnDCE, a transporter that in other bacteria mediates the uptake of alternative phosphorus sources. We previously reported that the transcriptional regulator PhnF controls the production of the Phn system, acting as a repressor under high-phosphate conditions. Here we show that the phnDCE genes are common among environmental mycobacteria, where they are often associated with phnF-like genes. In contrast, pathogenic mycobacteria were not found to encode Phn-like systems but instead were found to possess multiple copies of the pst genes. A detailed biochemical analysis of PhnF binding to its identified binding sites in the phnD-phnF intergenic region of M. smegmatis has allowed us to propose a quantitative model for repressor binding, which shows that a PhnF dimer binds independently to each site. We present the crystal structure of M. smegmatis PhnF at 1.8-Å resolution, showing a homodimer with a helix-turn-helix N-terminal domain and a C-terminal domain with a UbiC transcription regulator-associated fold. The C-terminal domain crystallized with a bound sulfate ion instead of the so far unidentified physiological ligand, allowing the identification of residues involved in effector binding. Comparison of the positioning of the DNA binding domains in PhnF with that in homologous proteins suggests that its DNA binding activity is regulated via a conformational change in the linker region, triggering a movement of the N-terminal domains. PMID:25049090

Dual Role of OhrR as a Repressor and an Activator in Response to Organic Hydroperoxides in Streptomyces coelicolor▿

PubMed Central

Oh, So-Young; Shin, Jung-Ho; Roe, Jung-Hye

2007-01-01

Organic hydroperoxide resistance in bacteria is achieved primarily through reducing oxidized membrane lipids. The soil-inhabiting aerobic bacterium Streptomyces coelicolor contains three paralogous genes for organic hydroperoxide resistance: ohrA, ohrB, and ohrC. The ohrA gene is transcribed divergently from ohrR, which encodes a putative regulator of MarR family. Both the ohrA and ohrR genes were induced highly by various organic hydroperoxides. The ohrA gene was induced through removal of repression by OhrR, whereas the ohrR gene was induced through activation by OhrR. Reduced OhrR bound to the ohrA-ohrR intergenic region, which contains a central (primary) and two adjacent (secondary) inverted-repeat motifs that overlap with promoter elements. Organic peroxide decreased the binding affinity of OhrR for the primary site, with a concomitant decrease in cooperative binding to the adjacent secondary sites. The single cysteine C28 in OhrR was involved in sensing oxidants, as determined by substitution mutagenesis. The C28S mutant of OhrR bound to the intergenic region without any change in binding affinity in response to organic peroxides. These results lead us to propose a model for the dual action of OhrR as a repressor and an activator in S. coelicolor. Under reduced conditions, OhrR binds cooperatively to the intergenic region, repressing transcription from both genes. Upon oxidation, the binding affinity of OhrR decreases, with a concomitant loss of cooperative binding, which allows RNA polymerase to bind to both the ohrA and ohrR promoters. The loosely bound oxidized OhrR can further activate transcription from the ohrR promoter. PMID:17586628

Comparative genomics of bacterial zinc regulons: enhanced ion transport, pathogenesis, and rearrangement of ribosomal proteins.

PubMed

Panina, Ekaterina M; Mironov, Andrey A; Gelfand, Mikhail S

2003-08-19

Zinc is an important component of many proteins, but in large concentrations it is poisonous to the cell. Thus its transport is regulated by zinc repressors ZUR of proteobacteria and Gram-positive bacteria from the Bacillus group and AdcR of bacteria from the Streptococcus group. Comparative computational analysis allowed us to identify binding signals of ZUR repressors GAAATGTTATANTATAACATTTC for gamma-proteobacteria, GTAATGTAATAACATTAC for the Agrobacterium group, GATATGTTATAACATATC for the Rhododoccus group, TAAATCGTAATNATTACGATTTA for Gram-positive bacteria, and TTAACYRGTTAA of the streptococcal AdcR repressor. In addition to known transporters and their paralogs, zinc regulons were predicted to contain a candidate component of the ATP binding cassette, zinT (b1995 in Escherichia coli and yrpE in Bacillus subtilis). Candidate AdcR-binding sites were identified upstream of genes encoding pneumococcal histidine triad (PHT) proteins from a number of pathogenic streptococci. Protein functional analysis of this family suggests that PHT proteins are involved in the invasion process. Finally, repression by zinc was predicted for genes encoding a variety of paralogs of ribosomal proteins. The original copies of all these proteins contain zinc-ribbon motifs and thus likely bind zinc, whereas these motifs are destroyed in zinc-regulated paralogs. We suggest that the induction of these paralogs in conditions of zinc starvation leads to their incorporation in a fraction of ribosomes instead of the original ribosomal proteins; the latter are then degraded with subsequent release of some zinc for the utilization by other proteins. Thus we predict a mechanism for maintaining zinc availability for essential enzymes.

Rce1, a novel transcriptional repressor, regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei.

PubMed

Cao, Yanli; Zheng, Fanglin; Wang, Lei; Zhao, Guolei; Chen, Guanjun; Zhang, Weixin; Liu, Weifeng

2017-07-01

Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1-mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1-encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy-efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei. © 2017 John Wiley & Sons Ltd.

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm

PubMed Central

Elwell, Jennifer A.; Lovato, TyAnna L.; Adams, Melanie M.; Baca, Erica M.; Lee, Thai; Cripps, Richard M.

2015-01-01

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arise through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist expression in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. PMID:25704510

The basic helix-loop-helix region of the transcriptional repressor hairy and enhancer of split 1 is preorganized to bind DNA.

PubMed

Popovic, Matija; Wienk, Hans; Coglievina, Maristella; Boelens, Rolf; Pongor, Sándor; Pintar, Alessandro

2014-04-01

Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions. Copyright © 2014 Wiley Periodicals, Inc.

kdgREcc Negatively Regulates Genes for Pectinases, Cellulase, Protease, HarpinEcc, and a Global RNA Regulator in Erwinia carotovora subsp. carotovora†

PubMed Central

Liu, Yang; Jiang, Guoqiao; Cui, Yaya; Mukherjee, Asita; Ma, Wei Lei; Chatterjee, Arun K.

1999-01-01

Erwinia carotovora subsp. carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces HarpinEcc, the elicitor of the hypersensitive reaction. We document here that KdgREcc (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgREch and the Escherichia coli repressor, KdgREco, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and HarpinEcc production in E. carotovora subsp. carotovora. The levels of pel-1, peh-1, celV, and hrpNEcc transcripts are markedly affected by KdgREcc. The KdgREcc− mutant is more virulent than the KdgREcc+ parent. Thus, our data for the first time establish a global regulatory role for KdgREcc in E. carotovora subsp. carotovora. Another novel observation is the negative effect of KdgREcc on the transcription of rsmB (previously aepH), which specifies an RNA regulator controlling exoenzyme and HarpinEcc production. The levels of rsmB RNA are higher in the KdgREcc− mutant than in the KdgREcc+ parent. Moreover, by DNase I protection assays we determined that purified KdgREcc protected three 25-bp regions within the transcriptional unit of rsmB. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgREcc-binding site as 5′-G/AA/TA/TGAAA[N6]TTTCAG/TG/TA-3′. Two such KdgREcc-binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgREcc-binding site within nucleotides +207 and +231. Analysis of lacZ transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of rsmB. KdgREcc also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lacZ transcriptional fusions. We conclude that KdgREcc affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgREcc affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription. PMID:10198003

The influence of repressor DNA binding site architecture on transcriptional control.

PubMed

Park, Dan M; Kiley, Patricia J

2014-08-26

How the architecture of DNA binding sites dictates the extent of repression of promoters is not well understood. Here, we addressed the importance of the number and information content of the three direct repeats (DRs) in the binding and repression of the icdA promoter by the phosphorylated form of the global Escherichia coli repressor ArcA (ArcA-P). We show that decreasing the information content of the two sites with the highest information (DR1 and DR2) eliminated ArcA binding to all three DRs and ArcA repression of icdA. Unexpectedly, we also found that DR3 occupancy functions principally in repression, since mutation of this low-information-content site both eliminated DNA binding to DR3 and significantly weakened icdA repression, despite the fact that binding to DR1 and DR2 was intact. In addition, increasing the information content of any one of the three DRs or addition of a fourth DR increased ArcA-dependent repression but perturbed signal-dependent regulation of repression. Thus, our data show that the information content and number of DR elements are critical architectural features for maintaining a balance between high-affinity binding and signal-dependent regulation of icdA promoter function in response to changes in ArcA-P levels. Optimization of such architectural features may be a common strategy to either dampen or enhance the sensitivity of DNA binding among the members of the large OmpR/PhoB family of regulators as well as other transcription factors. In Escherichia coli, the response regulator ArcA maintains homeostasis of redox carriers under O2-limiting conditions through a comprehensive repression of carbon oxidation pathways that require aerobic respiration to recycle redox carriers. Although a binding site architecture comprised of a variable number of sequence recognition elements has been identified within the promoter regions of ArcA-repressed operons, it is unclear how this variable architecture dictates transcriptional regulation. By dissecting the role of multiple sequence elements within the icdA promoter, we provide insight into the design principles that allow ArcA to repress transcription within diverse promoter contexts. Our data suggest that the arrangement of recognition elements is tailored to achieve sufficient repression of a given promoter while maintaining appropriate signal-dependent regulation of repression, providing insight into how diverse binding site architectures link changes in O2 with the fine-tuning of carbon oxidation pathway levels. Copyright © 2014 Park and Kiley.

Strong minor groove base conservation in sequence logos implies DNA distortion or base flipping during replication and transcription initiation | Center for Cancer Research

Cancer.gov

Dubbed "Tom's T" by Dhruba Chattoraj, the unusually conserved thymine at position +7 in bacteriophage P1 plasmid RepA DNA binding sites rises above repressor and acceptor sequence logos. The T appears to represent base flipping prior to helix opening in this DNA replication initation protein.

Mxi1 is a repressor of the c-Myc promoter and reverses activation by USF.

PubMed

Lee, T C; Ziff, E B

1999-01-08

The basic region/helix-loop-helix/leucine zipper (B-HLH-LZ) oncoprotein c-Myc is abundant in proliferating cells and forms heterodimers with Max protein that bind to E-box sites in DNA and stimulate genes required for proliferation. A second B-HLH-LZ protein, Mxi1, is induced during terminal differentiation, and forms heterodimers with Max that also bind E-boxes but tether the mSin3 transcriptional repressor protein along with histone deacetylase thereby antagonizing Myc-dependent activation. We show that Mxi1 also antagonizes Myc by a second pathway, repression of transcription from the major c-myc promoter, P2. Repression was independent of Mxi1 binding to mSin3 but dependent on the Mxi1 LZ and COOH-terminal sequences, including putative casein kinase II phosphorylation sites. Repression targeted elements of the myc P2 promoter core (-35/+10), where it reversed transactivation by the constitutive transcription factor, USF. We show that Zn2+ induction of a stably transfected, metallothionein promoter-regulated mxi1 gene blocked the ability of serum to induce transcription of the endogenous c-myc gene and cell entry into S phase. Thus, induction of Mxi1 in terminally differentiating cells may block Myc function by repressing the c-myc gene P2 promoter, as well as by antagonizing Myc-dependent transactivation through E-boxes.

The Zinc Finger Proteins Mxr1p and Repressor of Phosphoenolpyruvate Carboxykinase (ROP) Have the Same DNA Binding Specificity but Regulate Methanol Metabolism Antagonistically in Pichia pastoris*

PubMed Central

Kumar, Nallani Vijay; Rangarajan, Pundi N.

2012-01-01

The methanol-inducible alcohol oxidase I (AOXI) promoter of the methylotrophic yeast, Pichia pastoris, is used widely for the production of recombinant proteins. AOXI transcription is regulated by the zinc finger protein Mxr1p (methanol expression regulator 1). ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) is a methanol- and biotin starvation-inducible zinc finger protein that acts as a negative regulator of PEPCK in P. pastoris cultured in biotin-deficient, glucose-ammonium medium. The function of ROP during methanol metabolism is not known. In this study, we demonstrate that ROP represses methanol-inducible expression of AOXI when P. pastoris is cultured in a nutrient-rich medium containing yeast extract, peptone, and methanol (YPM). Deletion of the gene encoding ROP results in enhanced expression of AOXI and growth promotion whereas overexpression of ROP results in repression of AOXI and growth retardation of P. pastoris cultured in YPM medium. Surprisingly, deletion or overexpression of ROP has no effect on AOXI gene expression and growth of P. pastoris cultured in a minimal medium containing yeast nitrogen base and methanol (YNBM). Subcellular localization studies indicate that ROP translocates from cytosol to nucleus of cells cultured in YPM but not YNBM. In vitro DNA binding studies indicate that AOXI promoter sequences containing 5′ CYCCNY 3′ motifs serve as binding sites for Mxr1p as well as ROP. Thus, Mxr1p and ROP exhibit the same DNA binding specificity but regulate methanol metabolism antagonistically in P. pastoris. This is the first report on the identification of a transcriptional repressor of methanol metabolism in any yeast species. PMID:22888024

Borrelia oxidative stress response regulator, BosR: A distinctive Zn-dependent transcriptional activator

PubMed Central

Boylan, Julie A.; Posey, James E.; Gherardini, Frank C.

2003-01-01

The ability of a pathogen to cause infection depends on successful colonization of the host, which, in turn, requires adaptation to various challenges presented by that host. For example, host immune cells use a variety of mechanisms to control infection by bacterial pathogens, including the production of bactericidal reactive oxygen species. Prokaryotic and eukaryotic cells have developed ways of protecting themselves against this oxidative damage; for instance, Borrelia burgdorferi alters the expression of oxidative-stress-related proteins, such as a Dps/Dpr homolog NapA (BB0690), in response to increasing levels of oxygen and reactive oxygen species. These stress-related genes appear to be regulated by a putative metal-dependent DNA-binding protein (BB0647) that has 50.7% similarity to the peroxide-specific stress response repressor of Bacillus subtilis, PerR. We overexpressed and purified this protein from Escherichia coli and designated it Borrelia oxidative stress regulator, BosR. BosR bound to a 50-nt region 180 bp upstream of the napA transcriptional start site and required DTT and Zn2+ for optimal binding. Unlike the Bacillus subtilis PerR repressor, BosR did not require Fe2+ and Mn2+ for binding, and oxidizing agents, such as t-butyl peroxide, enhanced, not eliminated, BosR binding to the napA promoter region. Surprisingly, transcriptional fusion analysis indicated that BosR exerted a positive regulatory effect on napA that is inducible with t-butyl peroxide. On the basis of these data, we propose that, despite the similarity to PerR, BosR functions primarily as a transcriptional activator, not a repressor of oxidative stress response, in B. burgdorferi. PMID:12975527

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

PubMed Central

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

2016-01-01

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

Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

PubMed

Cruickshank, Mark N; Dods, James; Taylor, Rhonda L; Karimi, Mahdad; Fenwick, Emily J; Quail, Elizabeth A; Rea, Alexander J; Holers, V Michael; Abraham, Lawrence J; Ulgiati, Daniela

2015-07-01

Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12.

PubMed

Sellars, Laura E; Bryant, Jack A; Sánchez-Romero, María-Antonia; Sánchez-Morán, Eugenio; Busby, Stephen J W; Lee, David J

2017-08-03

In bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome. We developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication. Regions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm.

PubMed

Elwell, Jennifer A; Lovato, TyAnna L; Adams, Melanie M; Baca, Erica M; Lee, Thai; Cripps, Richard M

2015-04-15

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arises through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. Copyright © 2015 Elsevier Inc. All rights reserved.

Chorion gene activation and repression is dependent on BmC/EBP expression and binding to cognate cis-elements.

PubMed

Papantonis, Argyris; Sourmeli, Sissy; Lecanidou, Rena

2008-05-09

From the different cis-elements clustered on silkmoth chorion gene promoters, C/EBP binding sites predominate. Their sequence composition and dispersal vary amongst promoters of diverse developmental specificity. Occupancy of these sites by BmC/EBP was examined through Southwestern and ChIP assays modified to suit ovarian follicular cells. For the genes studied, binding of BmC/EBP coincided with the respective stages of transcriptional activation. However, the factor was reloaded on promoter sequences long after individual gene repression. Furthermore, suppression of BmC/EBP transcription in developing follicles resulted in de-regulation of chorion gene expression. A biphasic function of BmC/EBP, according to which it may act as both an activator and a repressor during silkmoth choriogenesis, is considered under the light of the presented data.

Structural basis for corepressor assembly by the orphan nuclear receptor TLX

PubMed Central

Zhou, X. Edward; He, Yuanzheng; Searose-Xu, Kelvin; Zhang, Chun-Li; Tsai, Chih-Cheng; Melcher, Karsten

2015-01-01

The orphan nuclear receptor TLX regulates neural stem cell self-renewal in the adult brain and functions primarily as a transcription repressor through recruitment of Atrophin corepressors, which bind to TLX via a conserved peptide motif termed the Atro box. Here we report crystal structures of the human and insect TLX ligand-binding domain in complex with Atro box peptides. In these structures, TLX adopts an autorepressed conformation in which its helix H12 occupies the coactivator-binding groove. Unexpectedly, H12 in this autorepressed conformation forms a novel binding pocket with residues from helix H3 that accommodates a short helix formed by the conserved ALXXLXXY motif of the Atro box. Mutations that weaken the TLX–Atrophin interaction compromise the repressive activity of TLX, demonstrating that this interaction is required for Atrophin to confer repressor activity to TLX. Moreover, the autorepressed conformation is conserved in the repressor class of orphan nuclear receptors, and mutations of corresponding residues in other members of this class of receptors diminish their repressor activities. Together, our results establish the functional conservation of the autorepressed conformation and define a key sequence motif in the Atro box that is essential for TLX-mediated repression. PMID:25691470

Structural basis for corepressor assembly by the orphan nuclear receptor TLX

DOE PAGES

Zhi, Xiaoyong; Zhou, X. Edward; He, Yuanzheng; ...

2015-02-15

The orphan nuclear receptor TLX regulates neural stem cell self-renewal in the adult brain and functions primarily as a transcription repressor through recruitment of Atrophin corepressors, which bind to TLX via a conserved peptide motif termed the Atro box. Here we report crystal structures of the human and insect TLX ligand-binding domain in complex with Atro box peptides. In these structures, TLX adopts an autorepressed conformation in which its helix H12 occupies the coactivator-binding groove. Unexpectedly, H12 in this autorepressed conformation forms a novel binding pocket with residues from helix H3 that accommodates a short helix formed by the conservedmore » ALXXLXXY motif of the Atro box. Mutations that weaken the TLX–Atrophin interaction compromise the repressive activity of TLX, demonstrating that this interaction is required for Atrophin to confer repressor activity to TLX. Moreover, the autorepressed conformation is conserved in the repressor class of orphan nuclear receptors, and mutations of corresponding residues in other members of this class of receptors diminish their repressor activities. Together, our results establish the functional conservation of the autorepressed conformation and define a key sequence motif in the Atro box that is essential for TLX-mediated repression.« less

Characterization of BreR Interaction with the Bile Response Promoters breAB and breR in Vibrio cholerae

PubMed Central

Cerda-Maira, Francisca A.; Kovacikova, Gabriela; Jude, Brooke A.; Skorupski, Karen

2013-01-01

The Vibrio cholerae BreR protein is a transcriptional repressor of the breAB efflux system operon, which encodes proteins involved in bile resistance. In a previous study (F. A. Cerda-Maira, C. S. Ringelberg, and R. K. Taylor, J. Bacteriol. 190:7441–7452, 2008), we used gel mobility shift assays to determine that BreR binds at two independent binding sites at the breAB promoter and a single site at its own promoter. Here it is shown, by DNase I footprinting and site-directed mutagenesis, that BreR is able to bind at a distal and a proximal site in the breAB promoter. However, only one of these sites, the proximal 29-bp site, is necessary for BreR-mediated transcriptional repression of breAB expression. In addition, it was determined that BreR represses its own expression by recognizing a 28-bp site at the breR promoter. These sites comprise regions of dyad symmetry within which residues critical for BreR function could be identified. The BreR consensus sequence AANGTANAC-N6-GTNTACNTT overlaps the −35 region at both promoters, implying that the repression of gene expression is achieved by interfering with RNA polymerase binding at these promoters. PMID:23144245

Architecture of a Fur Binding Site: a Comparative Analysis

PubMed Central

Lavrrar, Jennifer L.; McIntosh, Mark A.

2003-01-01

Fur is an iron-binding transcriptional repressor that recognizes a 19-bp consensus site of the sequence 5′-GATAATGATAATCATTATC-3′. This site can be defined as three adjacent hexamers of the sequence 5′-GATAAT-3′, with the third being slightly imperfect (an F-F-F configuration), or as two hexamers in the forward orientation separated by one base pair from a third hexamer in the reverse orientation (an F-F-x-R configuration). Although Fur can bind synthetic DNA sequences containing the F-F-F arrangement, most natural binding sites are variations of the F-F-x-R arrangement. The studies presented here compared the ability of Fur to recognize synthetic DNA sequences containing two to four adjacent hexamers with binding to sequences containing variations of the F-F-x-R arrangement (including natural operator sequences from the entS and fepB promoter regions of Escherichia coli). Gel retardation assays showed that the F-F-x-R architecture was necessary for high-affinity Fur-DNA interactions and that contiguous hexamers were not recognized as effectively. In addition, the stoichiometry of Fur at each binding site was determined, showing that Fur interacted with its minimal 19-bp binding site as two overlapping dimers. These data confirm the proposed overlapping-dimer binding model, where the unit of interaction with a single Fur dimer is two inverted hexamers separated by a C:G base pair, with two overlapping units comprising the 19-bp consensus binding site required for the high-affinity interaction with two Fur dimers. PMID:12644489

Genome-wide mapping and analysis of aryl hydrocarbon receptor (AHR)- and aryl hydrocarbon receptor repressor (AHRR)-binding sites in human breast cancer cells.

PubMed

Yang, Sunny Y; Ahmed, Shaimaa; Satheesh, Somisetty V; Matthews, Jason

2018-01-01

The aryl hydrocarbon receptor (AHR) mediates the toxic actions of environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), and also plays roles in vascular development, the immune response, and cell cycle regulation. The AHR repressor (AHRR) is an AHR-regulated gene and a negative regulator of AHR; however, the mechanisms of AHRR-dependent repression of AHR are unclear. In this study, we compared the genome-wide binding profiles of AHR and AHRR in MCF-7 human breast cancer cells treated for 24 h with TCDD using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). We identified 3915 AHR- and 2811 AHRR-bound regions, of which 974 (35%) were common to both datasets. When these 24-h datasets were also compared with AHR-bound regions identified after 45 min of TCDD treatment, 67% (1884) of AHRR-bound regions overlapped with those of AHR. This analysis identified 994 unique AHRR-bound regions. AHRR-bound regions mapped closer to promoter regions when compared with AHR-bound regions. The AHRE was identified and overrepresented in AHR:AHRR-co-bound regions, AHR-only regions, and AHRR-only regions. Candidate unique AHR- and AHRR-bound regions were validated by ChIP-qPCR and their ability to regulate gene expression was confirmed by luciferase reporter gene assays. Overall, this study reveals that AHR and AHRR exhibit similar but also distinct genome-wide binding profiles, supporting the notion that AHRR is a context- and gene-specific repressor of AHR activity.

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.

Regulation of the alpha-glucuronidase-encoding gene ( aguA) from Aspergillus niger.

PubMed

de Vries, R P; van de Vondervoort, P J I; Hendriks, L; van de Belt, M; Visser, J

2002-09-01

The alpha-glucuronidase gene aguA from Aspergillus niger was cloned and characterised. Analysis of the promoter region of aguA revealed the presence of four putative binding sites for the major carbon catabolite repressor protein CREA and one putative binding site for the transcriptional activator XLNR. In addition, a sequence motif was detected which differed only in the last nucleotide from the XLNR consensus site. A construct in which part of the aguA coding region was deleted still resulted in production of a stable mRNA upon transformation of A. niger. The putative XLNR binding sites and two of the putative CREA binding sites were mutated individually in this construct and the effects on expression were examined in A. niger transformants. Northern analysis of the transformants revealed that the consensus XLNR site is not actually functional in the aguA promoter, whereas the sequence that diverges from the consensus at a single position is functional. This indicates that XLNR is also able to bind to the sequence GGCTAG, and the XLNR binding site consensus should therefore be changed to GGCTAR. Both CREA sites are functional, indicating that CREA has a strong influence on aguA expression. A detailed expression analysis of aguA in four genetic backgrounds revealed a second regulatory system involved in activation of aguA gene expression. This system responds to the presence of glucuronic and galacturonic acids, and is not dependent on XLNR.

Proto-oncogene FBI-1 (Pokemon/ZBTB7A) represses transcription of the tumor suppressor Rb gene via binding competition with Sp1 and recruitment of co-repressors.

PubMed

Jeon, Bu-Nam; Yoo, Jung-Yoon; Choi, Won-Il; Lee, Choong-Eun; Yoon, Ho-Geun; Hur, Man-Wook

2008-11-28

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp -308 to -188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp -65 to -56) and GC-box 2 (bp -18 to -9), the latter of which is also bound by FBI-1. We found that FRE3 (bp -244 to -236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression.

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

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

Manjasetty, Babu A.; Halavaty, Andrei S.; Luan, Chi-Hao

Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription factors. The crystal structure of dimeric AcrR was determined and refined to 1.56 Å resolution. The tertiary and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether molecule bound. Residues from helices a4 and a7 shape the entry into this binding site. The structure of AcrR reveals that the extended helical conformation of helixmore » a4 is stabilized by the hydrogen bond between Glu67 (helix a4) and Gln130 (helix a7). Based on the structural comparison with the closest homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for control of the loop-to-helix transition within helix a4. This local conformational switch of helix a4 may be a key step in accessing the multidrug binding site and securing ligands at the binding site. Solution smallmolecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling the tetracyclic ring of cholesterol.« less

The rcsA Promoter of Pantoea stewartii subsp. stewartii Features a Low-Level Constitutive Promoter and an EsaR Quorum-Sensing-Regulated Promoter

PubMed Central

Carlier, Aurelien L.; von Bodman, S. B.

2006-01-01

The upstream region of the Pantoea stewartii rcsA gene features two promoters, one for constitutive basal-level expression and a second autoregulated promoter for induced expression. The EsaR quorum-sensing repressor binds to a site centered between the two promoters, blocking transcription elongation from the regulated promoter under noninducing conditions. PMID:16740966

Carbon repression of cellobiose dehydrogenase production in the white rot fungus Trametes versicolor is mediated at the level of gene transcription.

PubMed

Stapleton, P C; Dobson, A D W

2003-04-25

Cellobiose dehydrogenase (CDH) production in Trametes versicolor is induced in the presence of cellulose, but decreases when additional carbon sources such as glucose and maltose are added to the fungal cultures. Using T. versicolor-specific cdh primers in a reverse transcription-polymerase chain reaction-based approach, it appears that this repression in CDH production is being mediated at the level of gene transcription. When a 1.6-kb upstream region of the T. versicolor cdh gene was cloned and sequenced, a number of putative CreA-like binding sites were observed. We propose that these sites may be involved in mediating this repressive effect, based on their similarity to the consensus [5'-SYGGRGG-3'] site for binding of the CreA and Cre1 repressor proteins.

Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

NASA Astrophysics Data System (ADS)

Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

2015-07-01

Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

Escherichia coli ArgR mutants defective in cer/Xer recombination, but not in DNA binding.

PubMed

Sénéchal, Hélène; Delesques, Jérémy; Szatmari, George

2010-04-01

The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls the expression of the arginine biosynthetic genes and is required as an accessory factor for Xer site-specific recombination at cer and related recombination sites in plasmids. We used the technique of pentapeptide scanning mutagenesis to isolate a series of ArgR mutants that were considerably reduced in cer recombination, but were still able to repress an argA::lacZ fusion. DNA sequence analysis showed that all of the mutants mapped to the same nucleotide, resulting in a five amino acid insertion between residues 149 and 150 of ArgR, corresponding to the end of the alpha6 helix. A truncated ArgR containing a stop codon at residue 150 displayed the same phenotype as the protein with the five amino acid insertion, and both mutants displayed sequence-specific DNA-binding activity that was L-arginine dependent. These results show that the C-terminus of ArgR is more important in cer/Xer site-specific recombination than in DNA binding.

In vivo interference with AtTCP20 function induces severe plant growth alterations and deregulates the expression of many genes important for development.

PubMed

Hervé, Christine; Dabos, Patrick; Bardet, Claude; Jauneau, Alain; Auriac, Marie Christine; Ramboer, Agnès; Lacout, Fabrice; Tremousaygue, Dominique

2009-03-01

AtTCP20 is a transcription factor belonging to the Arabidopsis (Arabidopsis thaliana) TCP-P subfamily, characterized by its capacity to bind to site II motifs (TGGGCY). Our aim was to understand the role of AtTCP20 in plant development. The expression pattern of a translational fusion of Prom(TCP20):CDS20GUSGFP suggested a function for AtTCP20 in several plant organs and stages of development. The role of AtTCP20 was challenged in planta by inducing expression of AtTCP20 proteins fused with either a transcriptional activator domain (VP16) or a repressor domain (EAR). Expression of both modified proteins led to severe developmental phenotypes. In-depth analysis suggested that AtTCP20 may participate in the regulation of cell expansion, cell division, and cell differentiation. Gene expression profiling in roots and hypocotyls revealed that 252 genes were down-regulated in both organs after induction of the AtTCP20EAR repressor gene. Site II motifs (TGGGCY) were underrepresented in their promoters. Conversely, GG(A/T)CCC sequences related to binding sites identified for TCP proteins in rice (Oryza sativa) were overrepresented, and a TCP20 fusion protein was shown to bind to these sequences in vitro. Gene ontology indicated that many targeted genes were involved in cell wall biogenesis and modification during expansion and also encoded numerous transcription factors controlling plant development. Our results are consistent with the previous proposal that AtTCP20 is involved in cell division and growth coordination. Moreover, they further suggest that AtTCP20 also contributes to cell expansion control and indicate a different involvement of this protein in plant morphogenesis depending on the organ and the developmental stage.

Role of sequence encoded κB DNA geometry in gene regulation by Dorsal

PubMed Central

Mrinal, Nirotpal; Tomar, Archana; Nagaraju, Javaregowda

2011-01-01

Many proteins of the Rel family can act as both transcriptional activators and repressors. However, mechanism that discerns the ‘activator/repressor’ functions of Rel-proteins such as Dorsal (Drosophila homologue of mammalian NFκB) is not understood. Using genomic, biophysical and biochemical approaches, we demonstrate that the underlying principle of this functional specificity lies in the ‘sequence-encoded structure’ of the κB-DNA. We show that Dorsal-binding motifs exist in distinct activator and repressor conformations. Molecular dynamics of DNA-Dorsal complexes revealed that repressor κB-motifs typically have A-tract and flexible conformation that facilitates interaction with co-repressors. Deformable structure of repressor motifs, is due to changes in the hydrogen bonding in A:T pair in the ‘A-tract’ core. The sixth nucleotide in the nonameric κB-motif, ‘A’ (A6) in the repressor motifs and ‘T’ (T6) in the activator motifs, is critical to confer this functional specificity as A6 → T6 mutation transformed flexible repressor conformation into a rigid activator conformation. These results highlight that ‘sequence encoded κB DNA-geometry’ regulates gene expression by exerting allosteric effect on binding of Rel proteins which in turn regulates interaction with co-regulators. Further, we identified and characterized putative repressor motifs in Dl-target genes, which can potentially aid in functional annotation of Dorsal gene regulatory network. PMID:21890896

Structural basis for corepressor assembly by the orphan nuclear receptor TLX.

PubMed

Zhi, Xiaoyong; Zhou, X Edward; He, Yuanzheng; Searose-Xu, Kelvin; Zhang, Chun-Li; Tsai, Chih-Cheng; Melcher, Karsten; Xu, H Eric

2015-02-15

The orphan nuclear receptor TLX regulates neural stem cell self-renewal in the adult brain and functions primarily as a transcription repressor through recruitment of Atrophin corepressors, which bind to TLX via a conserved peptide motif termed the Atro box. Here we report crystal structures of the human and insect TLX ligand-binding domain in complex with Atro box peptides. In these structures, TLX adopts an autorepressed conformation in which its helix H12 occupies the coactivator-binding groove. Unexpectedly, H12 in this autorepressed conformation forms a novel binding pocket with residues from helix H3 that accommodates a short helix formed by the conserved ALXXLXXY motif of the Atro box. Mutations that weaken the TLX-Atrophin interaction compromise the repressive activity of TLX, demonstrating that this interaction is required for Atrophin to confer repressor activity to TLX. Moreover, the autorepressed conformation is conserved in the repressor class of orphan nuclear receptors, and mutations of corresponding residues in other members of this class of receptors diminish their repressor activities. Together, our results establish the functional conservation of the autorepressed conformation and define a key sequence motif in the Atro box that is essential for TLX-mediated repression. © 2015 Zhi et al.; Published by Cold Spring Harbor Laboratory Press.

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

Loop-to-helix transition in the structure of multidrug regulator AcrR at the entrance of the drug-binding cavity.

PubMed

Manjasetty, Babu A; Halavaty, Andrei S; Luan, Chi-Hao; Osipiuk, Jerzy; Mulligan, Rory; Kwon, Keehwan; Anderson, Wayne F; Joachimiak, Andrzej

2016-04-01

Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription factors. The crystal structure of dimeric AcrR was determined and refined to 1.56Å resolution. The tertiary and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether molecule bound. Residues from helices α4 and α7 shape the entry into this binding site. The structure of AcrR reveals that the extended helical conformation of helix α4 is stabilized by the hydrogen bond between Glu67 (helix α4) and Gln130 (helix α7). Based on the structural comparison with the closest homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for control of the loop-to-helix transition within helix α4. This local conformational switch of helix α4 may be a key step in accessing the multidrug binding site and securing ligands at the binding site. Solution small-molecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling the tetracyclic ring of cholesterol. Copyright © 2016. Published by Elsevier Inc.

The Activation Domain of the Bovine Papillomavirus E2 Protein Mediates Association of DNA-Bound Dimers to form DNA Loops

NASA Astrophysics Data System (ADS)

Knight, Jonathan D.; Li, Rong; Botchan, Michael

1991-04-01

The E2 transactivator protein of bovine papillomavirus binds its specific DNA target sequence as a dimer. We have found that E2 dimers, performed in solution independent of DNA, exhibit substantial cooperativity of DNA binding as detected by both nitrocellulose filter retention and footprint analysis techniques. If the binding sites are widely spaced, E2 forms stable DNA loops visible by electron microscopy. When three widely separated binding sites reside on te DNA, E2 condenses the molecule into a bow-tie structure. This implies that each E2 dimer has at least two independent surfaces for multimerization. Two naturally occurring shorter forms of the protein, E2C and D8/E2, which function in vivo as repressors of transcription, do not form such loops. Thus, the looping function of E2 maps to the 161-amino acid activation domain. These results support the looping model of transcription activation by enhancers.

Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition.

PubMed

Madoori, Pramod Kumar; Agustiandari, Herfita; Driessen, Arnold J M; Thunnissen, Andy-Mark W H

2009-01-21

LmrR is a PadR-related transcriptional repressor that regulates the production of LmrCD, a major multidrug ABC transporter in Lactococcus lactis. Transcriptional regulation is presumed to follow a drug-sensitive induction mechanism involving the direct binding of transporter ligands to LmrR. Here, we present crystal structures of LmrR in an apo state and in two drug-bound states complexed with Hoechst 33342 and daunomycin. LmrR shows a common topology containing a typical beta-winged helix-turn-helix domain with an additional C-terminal helix involved in dimerization. Its dimeric organization is highly unusual with a flat-shaped hydrophobic pore at the dimer centre serving as a multidrug-binding site. The drugs bind in a similar manner with their aromatic rings sandwiched in between the indole groups of two dimer-related tryptophan residues. Multidrug recognition is facilitated by conformational plasticity and the absence of drug-specific hydrogen bonds. Combined analyses using site-directed mutagenesis, fluorescence-based drug binding and protein-DNA gel shift assays reveal an allosteric coupling between the multidrug- and DNA-binding sites of LmrR that most likely has a function in the induction mechanism.

SnoN co-repressor binds and represses smad7 gene promoter.

PubMed

Briones-Orta, Marco A; Sosa-Garrocho, Marcela; Moreno-Alvarez, Paola; Fonseca-Sánchez, Miguel A; Macías-Silva, Marina

2006-03-17

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.

The cAMP receptor protein CRP can function as an osmoregulator of transcription in Escherichia coli

PubMed Central

Landis, Lenore; Xu, Jimin; Johnson, Reid C.

1999-01-01

Transcription of the P1 promoter of the Escherichia coli proP gene, which encodes a transporter of osmoprotectants, is strongly induced by a shift to hyperosmotic media. Unlike most other osmotically regulated promoters, the induction occurs for a brief period of time, corresponding to the replacement of intracellular K+ glutamate with osmoprotecting compounds. This burst of proP transcription is correlated with the osmolarity-dependent binding of the cAMP receptor protein CRP to a site within the proP P1 promoter. We show that CRP–cAMP functions as an osmotically sensitive repressor of proP P1 transcription in vitro. Binding of CRP to the proP promoter in vivo is transiently destabilized after a hyperosmotic shift with kinetics that correspond to the derepression of transcription, whereas Fis and Lac repressor binding is not osmotically sensitive. Similar osmotic regulation of proP P1 transcription by the CRP* mutant implies that binding of cAMP is not responsible for the unusual osmotic sensitivity of CRP activity. Osmotic regulation of CRP activity is not limited to proP. Activation of the lac promoter by CRP is also transiently inhibited after an osmotic upshift, as is the binding of CRP to the galΔ4 P1 promoter. These findings suggest that CRP functions in certain contexts to regulate gene expression in response to osmotic changes, in addition to its role in catabolite control. PMID:10601034

The cAMP receptor protein CRP can function as an osmoregulator of transcription in Escherichia coli.

PubMed

Landis, L; Xu, J; Johnson, R C

1999-12-01

Transcription of the P1 promoter of the Escherichia coli proP gene, which encodes a transporter of osmoprotectants, is strongly induced by a shift to hyperosmotic media. Unlike most other osmotically regulated promoters, the induction occurs for a brief period of time, corresponding to the replacement of intracellular K(+) glutamate with osmoprotecting compounds. This burst of proP transcription is correlated with the osmolarity-dependent binding of the cAMP receptor protein CRP to a site within the proP P1 promoter. We show that CRP-cAMP functions as an osmotically sensitive repressor of proP P1 transcription in vitro. Binding of CRP to the proP promoter in vivo is transiently destabilized after a hyperosmotic shift with kinetics that correspond to the derepression of transcription, whereas Fis and Lac repressor binding is not osmotically sensitive. Similar osmotic regulation of proP P1 transcription by the CRP* mutant implies that binding of cAMP is not responsible for the unusual osmotic sensitivity of CRP activity. Osmotic regulation of CRP activity is not limited to proP. Activation of the lac promoter by CRP is also transiently inhibited after an osmotic upshift, as is the binding of CRP to the galdelta4P1 promoter. These findings suggest that CRP functions in certain contexts to regulate gene expression in response to osmotic changes, in addition to its role in catabolite control.

The biotin repressor: modulation of allostery by corepressor analogs.

PubMed

Brown, Patrick H; Cronan, John E; Grøtli, Morten; Beckett, Dorothy

2004-04-02

The Escherichia coli biotin repressor functions in biotin retention and regulation of biotin biosynthesis. Biotin retention is accomplished via the two-step biotinylation of the biotin-dependent enzyme, acetyl-CoA carboxylase. In the first step of this reaction the substrates biotin and ATP are utilized in synthesis of the activated biotin, biotinyl-5'-AMP, while in the second step this activated biotin is transferred to a unique lysine residue of the biotin carboxyl carrier protein subunit of the carboxylase. Regulation of biotin biosynthesis is accomplished through binding of the repressor to the transcription control region of the biotin biosynthetic operon. The adenylated or activated biotin functions as the corepressor in this DNA binding process. The activated biotin is a mixed anhydride and thus labile. In efforts to develop tools for structural and thermodynamic studies of the biotin regulatory interactions, two analogs of the adenylate, a sulfamoyl derivative and an ester derivative, have been synthesized and functionally characterized. Results of fluorescence measurements indicate that both analogs bind with high affinity to the repressor and that both are inactive in biotin transfer to the acceptor protein. Functional studies of their corepressor properties indicate that while the sulfamoyl is a weak allosteric activator, the ester closely mimics the physiological corepressor in activation of assembly of the transcription repression complex. Results of these studies also provide further insight into the allosteric mechanism of the biotin repressor.

Context-dependent EKLF responsiveness defines the developmental specificity of the human ɛ-globin gene in erythroid cells of YAC transgenic mice

PubMed Central

Tanimoto, Keiji; Liu, Qinghui; Grosveld, Frank; Bungert, Jörg; Engel, James Douglas

2000-01-01

We explored the mechanism of definitive-stage ɛ-globin transcriptional inactivity within a human β-globin YAC expressed in transgenic mice. We focused on the globin CAC and CAAT promoter motifs, as previous laboratory and clinical studies indicated a pivotal role for these elements in globin gene activation. A high-affinity CAC-binding site for the erythroid krüppel-like factor (EKLF) was placed in the ɛ-globin promoter at a position corresponding to that in the adult β-globin promoter, thereby simultaneously ablating a direct repeat (DR) element. This mutation led to EKLF-independent ɛ-globin transcription during definitive erythropoiesis. A second 4-bp substitution in the ɛ-globin CAAT sequence, which simultaneously disrupts a second DR element, further enhanced ectopic definitive erythroid activation of ɛ-globin transcription, which surprisingly became EKLF dependent. We finally examined factors in nuclear extracts prepared from embryonic or adult erythroid cells that bound these elements in vitro, and we identified a novel DR-binding protein (DRED) whose properties are consistent with those expected for a definitive-stage ɛ-globin repressor. We conclude that the suppression of ɛ-globin transcription during definitive erythropoiesis is mediated by the binding of a repressor that prevents EKLF from activating the ɛ-globin gene. PMID:11069894

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Transcription factor FBI-1 acts as a dual regulator in adipogenesis by coordinated regulation of cyclin-A and E2F-4.

PubMed

Laudes, Matthias; Bilkovski, Roman; Oberhauser, Frank; Droste, Andrea; Gomolka, Matthias; Leeser, Uschi; Udelhoven, Michael; Krone, Wilhelm

2008-05-01

Generation of new adipocytes plays a major role in the development of obesity. We previously have shown that transcriptional repressor factor that binds to IST (FBI)-1 exerts a dual effect in the process of adipogenesis by inhibiting proliferation and promoting differentiation of preadipocytes. The aim of the present study was to identify FBI-1 regulated molecular effectors that could account for these effects. Overexpressing FBI-1 in preadipocytes resulted in reduced expression of the cell cycle regulator cyclin A, which may explain FBI-1 induced inhibition of proliferation. Interestingly, FBI-1 repressed cyclin A promoter activity through an indirect mechanisms that did not involve direct binding of FBI-1 to the promoter sequence, but rather FBI-1 inhibition of transcriptional activator Sp1 binding to a regulatory element at -452 to -443. We also show that FBI-1 promotes terminal preadipocyte differentiation through a mechanism involving decreased levels of expression of the PPARgamma inhibitor E2F-4. FBI-1 significantly reduced E2F-4 promoter activity. Contrary to cyclin A, we found FBI-1-induced repression of E2F-4 is mediated by a direct mechanism via a FBI-1 regulatory element at -11 to -5. As function of transcriptional repressors normally depends on the presence of regulatory co-factors we also performed expression profiling of potential FBI-1 co-repressors throughout adipogenesis. In these experiments Sin3A and histon deacetylase (HDAC)-1 showed a similar expression pattern compared to FBI-1. Strikingly, co-immunoprecipitation studies revealed that FBI-1 binds Sin3A and HDAC-1 to form a repressor complex. Furthermore, by mutational analysis the amino terminal Poxvirus (POZ) domain of FBI-1 was found to be important for Sin3A and HDAC-1 binding. Taken together, FBI-1 is the first transcriptional repressor shown to act as a dual regulator in adipogenesis exerting repressor activities on target genes by both, direct and indirect mechanisms.

Proto-oncogene FBI-1 (Pokemon/ZBTB7A) Represses Transcription of the Tumor Suppressor Rb Gene via Binding Competition with Sp1 and Recruitment of Co-repressors*S⃞

PubMed Central

Jeon, Bu-Nam; Yoo, Jung-Yoon; Choi, Won-Il; Lee, Choong-Eun; Yoon, Ho-Geun; Hur, Man-Wook

2008-01-01

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp –308 to –188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp –65 to –56) and GC-box 2 (bp –18 to –9), the latter of which is also bound by FBI-1. We found that FRE3 (bp –244 to –236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression. PMID:18801742

Decreased Sensitivity to Changes in the Concentration of Metal Ions as the Basis for the Hyperactivity of DtxR(E175K)

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

D’Aquino, J. Alejandro; Denninger, Andrew R.; Moulin, Aaron G.

2010-01-12

The metal-ion-activated diphtheria toxin repressor (DtxR) is responsible for the regulation of virulence and other genes in Corynebacterium diphtheriae. A single point mutation in DtxR, DtxR(E175K), causes this mutant repressor to have a hyperactive phenotype. Mice infected with Mycobacterium tuberculosis transformed with plasmids carrying this mutant gene show reduced signs of the tuberculosis infection. Corynebacterial DtxR is able to complement mycobacterial IdeR and vice versa. To date, an explanation for the hyperactivity of DtxR(E175K) has remained elusive. In an attempt to address this issue, we have solved the first crystal structure of DtxR(E175K) and characterized this mutant using circular dichroism,more » isothermal titration calorimetry, and other biochemical techniques. The results show that although DtxR(E175K) and the wild type have similar secondary structures, DtxR(E175K) gains additional thermostability upon activation with metal ions, which may lead to this mutant requiring a lower concentration of metal ions to reach the same levels of thermostability as the wild-type protein. The E175K mutation causes binding site 1 to retain metal ion bound at all times, which can only be removed by incubation with an ion chelator. The crystal structure of DtxR(E175K) shows an empty binding site 2 without evidence of oxidation of Cys102. The association constant for this low-affinity binding site of DtxR(E175K) obtained from calorimetric titration with Ni(II) is K{sub a} = 7.6 {+-} 0.5 x 10{sup 4}, which is very similar to the reported value for the wild-type repressor, K{sub a} = 6.3 x 10{sup 4}. Both the wild type and DtxR(E175K) require the same amount of metal ion to produce a shift in the electrophoretic mobility shift assay, but unlike the wild type, DtxR(E175K) binding to its cognate DNA [tox promoter-operator (toxPO)] does not require metal-ion supplementation in the running buffer. In the timescale of these experiments, the Mn(II)-DtxR(E175K)-toxPO complex is insensitive to changes in the environmental cation concentrations. In addition to Mn(II), Ni(II), Co(II), Cd(II), and Zn(II) are able to sustain the hyperactive phenotype. These results demonstrate a prominent role of binding site 1 in the activation of DtxR and support the hypothesis that DtxR(E175K) attenuates the expression of virulence due to the decreased ability of the Me(II)-DtxR(E175K)-toxPO complex to dissociate at low concentrations of metal ions.« less

Regulation of CYBB Gene Expression in Human Phagocytes by a Distant Upstream NF-κB Binding Site.

PubMed

Frazão, Josias B; Thain, Alison; Zhu, Zhiqing; Luengo, Marcos; Condino-Neto, Antonio; Newburger, Peter E

2015-09-01

The human CYBB gene encodes the gp91-phox component of the phagocyte oxidase enzyme complex, which is responsible for generating superoxide and other downstream reactive oxygen species essential to microbial killing. In the present study, we have identified by sequence analysis a putative NF-κB binding site in a DNase I hypersensitive site, termed HS-II, located in the distant 5' flanking region of the CYBB gene. Electrophoretic mobility assays showed binding of the sequence element by recombinant NF-κB protein p50 and by proteins in nuclear extract from the HL-60 myeloid leukemia cell line corresponding to p50 and to p50/p65 heterodimers. Chromatin immunoprecipitation demonstrated NF-κB binding to the site in intact HL-60 cells. Chromosome conformation capture (3C) assays demonstrated physical interaction between the NF-κB binding site and the CYBB promoter region. Inhibition of NF-κB activity by salicylate reduced CYBB expression in peripheral blood neutrophils and differentiated U937 monocytic leukemia cells. U937 cells transfected with a mutant inhibitor of κB "super-repressor" showed markedly diminished CYBB expression. Luciferase reporter analysis of the NF-κB site linked to the CYBB 5' flanking promoter region revealed enhanced expression, augmented by treatment with interferon-γ. These studies indicate a role for this distant, 15 kb upstream, binding site in NF-κB regulation of the CYBB gene, an essential component of phagocyte-mediated host defense. © 2015 Wiley Periodicals, Inc.

Neisseria conserved protein DMP19 is a DNA mimic protein that prevents DNA binding to a hypothetical nitrogen-response transcription factor

PubMed Central

Wang, Hao-Ching; Ko, Tzu-Ping; Wu, Mao-Lun; Ku, Shan-Chi; Wu, Hsing-Ju; Wang, Andrew H.-J.

2012-01-01

DNA mimic proteins occupy the DNA binding sites of DNA-binding proteins, and prevent these sites from being accessed by DNA. We show here that the Neisseria conserved hypothetical protein DMP19 acts as a DNA mimic. The crystal structure of DMP19 shows a dsDNA-like negative charge distribution on the surface, suggesting that this protein should be added to the short list of known DNA mimic proteins. The crystal structure of another related protein, NHTF (Neisseria hypothetical transcription factor), provides evidence that it is a member of the xenobiotic-response element (XRE) family of transcriptional factors. NHTF binds to a palindromic DNA sequence containing a 5′-TGTNAN11TNACA-3′ recognition box that controls the expression of an NHTF-related operon in which the conserved nitrogen-response protein [i.e. (Protein-PII) uridylyltransferase] is encoded. The complementary surface charges between DMP19 and NHTF suggest specific charge–charge interaction. In a DNA-binding assay, we found that DMP19 can prevent NHTF from binding to its DNA-binding sites. Finally, we used an in situ gene regulation assay to provide evidence that NHTF is a repressor of its down-stream genes and that DMP19 can neutralize this effect. We therefore conclude that the interaction of DMP19 and NHTF provides a novel gene regulation mechanism in Neisseria spps. PMID:22373915

Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair

PubMed Central

Shinohara, Takeshi; Ikawa, Shukuko; Iwasaki, Wakana; Hiraki, Toshiki; Hikima, Takaaki; Mikawa, Tsutomu; Arai, Naoto; Kamiya, Nobuo; Shibata, Takehiko

2015-01-01

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions. PMID:25561575

Thermodynamics-Based Models of Transcriptional Regulation by Enhancers: The Roles of Synergistic Activation, Cooperative Binding and Short-Range Repression

PubMed Central

He, Xin; Samee, Md. Abul Hassan; Blatti, Charles; Sinha, Saurabh

2010-01-01

Quantitative models of cis-regulatory activity have the potential to improve our mechanistic understanding of transcriptional regulation. However, the few models available today have been based on simplistic assumptions about the sequences being modeled, or heuristic approximations of the underlying regulatory mechanisms. We have developed a thermodynamics-based model to predict gene expression driven by any DNA sequence, as a function of transcription factor concentrations and their DNA-binding specificities. It uses statistical thermodynamics theory to model not only protein-DNA interaction, but also the effect of DNA-bound activators and repressors on gene expression. In addition, the model incorporates mechanistic features such as synergistic effect of multiple activators, short range repression, and cooperativity in transcription factor-DNA binding, allowing us to systematically evaluate the significance of these features in the context of available expression data. Using this model on segmentation-related enhancers in Drosophila, we find that transcriptional synergy due to simultaneous action of multiple activators helps explain the data beyond what can be explained by cooperative DNA-binding alone. We find clear support for the phenomenon of short-range repression, where repressors do not directly interact with the basal transcriptional machinery. We also find that the binding sites contributing to an enhancer's function may not be conserved during evolution, and a noticeable fraction of these undergo lineage-specific changes. Our implementation of the model, called GEMSTAT, is the first publicly available program for simultaneously modeling the regulatory activities of a given set of sequences. PMID:20862354

The Pseudomonas aeruginosa Catabolite Repression Control Protein Crc Is Devoid of RNA Binding Activity

PubMed Central

Djinovic-Carugo, Kristina; Bläsi, Udo

2013-01-01

The Crc protein has been shown to mediate catabolite repression control in Pseudomonas, leading to a preferential assimilation of carbon sources. It has been suggested that Crc acts as a translational repressor of mRNAs, encoding functions involved in uptake and breakdown of different carbon sources. Moreover, the regulatory RNA CrcZ, the level of which is increased in the presence of less preferred carbon sources, was suggested to bind to and sequester Crc, resulting in a relief of catabolite repression. Here, we determined the crystal structure of Pseudomonas aeruginosa Crc, a member of apurinic/apyrimidinic (AP) endonuclease family, at 1.8 Å. Although Crc displays high sequence similarity with its orthologs, there are amino acid alterations in the area corresponding to the active site in AP proteins. Unlike typical AP endonuclease family proteins, Crc has a reduced overall positive charge and the conserved positively charged amino-acid residues of the DNA-binding surface of AP proteins are partially substituted by negatively charged, polar and hydrophobic residues. Crc protein purified to homogeneity from P. aeruginosa did neither display DNase activity, nor did it bind to previously identified RNA substrates. Rather, the RNA chaperone Hfq was identified as a contaminant in His-tagged Crc preparations purified by one step Ni-affinity chromatography from Escherichia coli, and was shown to account for the RNA binding activity observed with the His-Crc preparations. Taken together, these data challenge a role of Crc as a direct translational repressor in carbon catabolite repression in P. aeruginosa. PMID:23717639

The Pseudomonas aeruginosa catabolite repression control protein Crc is devoid of RNA binding activity.

PubMed

Milojevic, Tetyana; Grishkovskaya, Irina; Sonnleitner, Elisabeth; Djinovic-Carugo, Kristina; Bläsi, Udo

2013-01-01

The Crc protein has been shown to mediate catabolite repression control in Pseudomonas, leading to a preferential assimilation of carbon sources. It has been suggested that Crc acts as a translational repressor of mRNAs, encoding functions involved in uptake and breakdown of different carbon sources. Moreover, the regulatory RNA CrcZ, the level of which is increased in the presence of less preferred carbon sources, was suggested to bind to and sequester Crc, resulting in a relief of catabolite repression. Here, we determined the crystal structure of Pseudomonas aeruginosa Crc, a member of apurinic/apyrimidinic (AP) endonuclease family, at 1.8 Å. Although Crc displays high sequence similarity with its orthologs, there are amino acid alterations in the area corresponding to the active site in AP proteins. Unlike typical AP endonuclease family proteins, Crc has a reduced overall positive charge and the conserved positively charged amino-acid residues of the DNA-binding surface of AP proteins are partially substituted by negatively charged, polar and hydrophobic residues. Crc protein purified to homogeneity from P. aeruginosa did neither display DNase activity, nor did it bind to previously identified RNA substrates. Rather, the RNA chaperone Hfq was identified as a contaminant in His-tagged Crc preparations purified by one step Ni-affinity chromatography from Escherichia coli, and was shown to account for the RNA binding activity observed with the His-Crc preparations. Taken together, these data challenge a role of Crc as a direct translational repressor in carbon catabolite repression in P. aeruginosa.

GIGANTEA directly activates Flowering Locus T in Arabidopsis thaliana.

PubMed

Sawa, Mariko; Kay, Steve A

2011-07-12

Plants perceive environmental signals such as day length and temperature to determine optimal timing for the transition from vegetative to floral stages. Arabidopsis flowers under long-day conditions through the CONSTANS (CO)-FLOWERING LOCUS T (FT) regulatory module. It is thought that the environmental cues for photoperiodic control of flowering are initially perceived in the leaves. We have previously shown that GIGANTEA (GI) regulates the timing of CO expression, together with FLAVIN-BINDING, KELCH REPEAT, F BOX protein 1. Normally, CO and FT are expressed exclusively in vascular bundles, whereas GI is expressed in various tissues. To better elucidate the role of tissue-specific expression of GI in the flowering pathway, we established transgenic lines in which GI is expressed exclusively in mesophyll, vascular bundles, epidermis, shoot apical meristem, or root. We found that GI expressed in either mesophyll or vascular bundles rescues the late-flowering phenotype of the gi-2 loss-of-function mutant under both short-day and long-day conditions. Interestingly, GI expressed in mesophyll or vascular tissues increases FT expression without up-regulating CO expression under short-day conditions. Furthermore, we examined the interaction between GI and FT repressors in mesophyll. We found that GI can bind to three FT repressors: SHORT VEGETATIVE PHASE (SVP), TEMPRANILLO (TEM)1, and TEM2. Finally, our chromatin immunoprecipitation experiments showed that GI binds to FT promoter regions that are near the SVP binding sites. Taken together, our data further elucidate the multiple roles of GI in the regulation of flowering time.

A Small-Molecule Inhibitor of BCL6 Kills DLBCL Cells In Vitro and In Vivo

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

Cerchietti, L.C.; Ghetu, A.F.; Zhu, X.

2010-09-22

The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low-molecular-weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was nontoxic and potently suppressed DLBCL tumors in vivo. The compoundmore » also killed primary DLBCLs from human patients.« less

Binding of the Zn2+ ion to ferric uptake regulation protein from E. coli and the competition with Fe2+ binding: a molecular modeling study of the effect on DNA binding and conformational changes of Fur

NASA Astrophysics Data System (ADS)

Jabour, Salih; Hamed, Mazen Y.

2009-04-01

The three dimensional structure of Ferric uptake regulation protein dimer from E. coli, determined by molecular modeling, was docked on a DNA fragment (iron box) and Zn2+ ions were added in two steps. The first step involved the binding of one Zn2+ ion to what is known as the zinc site which consists of the residues Cys 92, Cys 95, Asp 137, Asp141, Arg139, Glu 140, His 145 and His 143 with an average metal-Nitrogen distance of 2.5 Å and metal-oxygen distance of 3.1-3.2 Å. The second Zn2+ ion is bound to the iron activating site formed from the residues Ile 50, His 71, Asn 72, Gly 97, Asp 105 and Ala 109. The binding of the second Zn2+ ion strengthened the binding of the first ion as indicated by the shortening of the zinc-residue distances. Fe2+, when added to the complex consisting of 2Zn2+/Fur dimer/DNA, replaced the Zn2+ ion in the zinc site and when a second Fe2+ was added, it replaced the second zinc ion in the iron activating site. The binding of both zinc and iron ions induced a similar change in Fur conformations, but shifted residues closer to DNA in a different manner. This is discussed along with a possible role for the Zn2+ ion in the Fur dimer binding of DNA in its repressor activity.

DNA residence time is a regulatory factor of transcription repression

PubMed Central

Clauß, Karen; Popp, Achim P.; Schulze, Lena; Hettich, Johannes; Reisser, Matthias; Escoter Torres, Laura; Uhlenhaut, N. Henriette

2017-01-01

Abstract Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation. PMID:28977492

Cryptic glucocorticoid receptor-binding sites pervade genomic NF-κB response elements.

PubMed

Hudson, William H; Vera, Ian Mitchelle S de; Nwachukwu, Jerome C; Weikum, Emily R; Herbst, Austin G; Yang, Qin; Bain, David L; Nettles, Kendall W; Kojetin, Douglas J; Ortlund, Eric A

2018-04-06

Glucocorticoids (GCs) are potent repressors of NF-κB activity, making them a preferred choice for treatment of inflammation-driven conditions. Despite the widespread use of GCs in the clinic, current models are inadequate to explain the role of the glucocorticoid receptor (GR) within this critical signaling pathway. GR binding directly to NF-κB itself-tethering in a DNA binding-independent manner-represents the standing model of how GCs inhibit NF-κB-driven transcription. We demonstrate that direct binding of GR to genomic NF-κB response elements (κBREs) mediates GR-driven repression of inflammatory gene expression. We report five crystal structures and solution NMR data of GR DBD-κBRE complexes, which reveal that GR recognizes a cryptic response element between the binding footprints of NF-κB subunits within κBREs. These cryptic sequences exhibit high sequence and functional conservation, suggesting that GR binding to κBREs is an evolutionarily conserved mechanism of controlling the inflammatory response.

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

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

Procházková, Kateřina; Čermáková, Kateřina; Pachl, Petr

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 similaritymore » 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.« less

Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin.

PubMed Central

Gumucio, D L; Rood, K L; Gray, T A; Riordan, M F; Sartor, C I; Collins, F S

1988-01-01

The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation. Images PMID:2468996

The insulator protein Suppressor of Hairy-wing is an essential transcriptional repressor in the Drosophila ovary.

PubMed

Soshnev, Alexey A; Baxley, Ryan M; Manak, J Robert; Tan, Kai; Geyer, Pamela K

2013-09-01

Suppressor of Hairy-wing [Su(Hw)] is a DNA-binding factor required for gypsy insulator function and female germline development in Drosophila. The insulator function of the gypsy retrotransposon depends on Su(Hw) binding to clustered Su(Hw) binding sites (SBSs) and recruitment of the insulator proteins Centrosomal Protein 190 kD (CP190) and Modifier of mdg4 67.2 kD (Mod67.2). By contrast, the Su(Hw) germline function involves binding to non-clustered SBSs and does not require CP190 or Mod67.2. Here, we identify Su(Hw) target genes, using genome-wide analyses in the ovary to uncover genes with an ovary-bound SBS that are misregulated upon Su(Hw) loss. Most Su(Hw) target genes demonstrate enriched expression in the wild-type CNS. Loss of Su(Hw) leads to increased expression of these CNS-enriched target genes in the ovary and other tissues, suggesting that Su(Hw) is a repressor of neural genes in non-neural tissues. Among the Su(Hw) target genes is RNA-binding protein 9 (Rbp9), a member of the ELAV/Hu gene family. Su(Hw) regulation of Rbp9 appears to be insulator independent, as Rbp9 expression is unchanged in a genetic background that compromises the functions of the CP190 and Mod67.2 insulator proteins, even though both localize to Rbp9 SBSs. Rbp9 misregulation is central to su(Hw)(-/-) sterility, as Rbp9(+/-), su(Hw)(-/-) females are fertile. Eggs produced by Rbp9(+/-), su(Hw)(-/-) females show patterning defects, revealing a somatic requirement for Su(Hw) in the ovary. Our studies demonstrate that Su(Hw) is a versatile transcriptional regulatory protein with an essential developmental function involving transcriptional repression.

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

DOE PAGES

Killikelly, April; Jakoncic, Jean; Benson, Meredith A.; ...

2014-10-20

Staphylococcus aureus is responsible for a large number of diverse infections worldwide. In order to support its pathogenic lifestyle, S. aureus has to regulate the expression of virulence factors in a coordinated fashion. One of the central regulators of the S. aureus virulence regulatory networks is the transcription factor repressor of toxin (Rot). Rot plays a key role in regulating S. aureus virulence through activation or repression of promoters that control expression of a large number of critical virulence factors. However, the mechanism by which Rot mediates gene regulation has remained elusive. Here, we have determined the crystal structure ofmore » Rot and used this information to probe the contribution made by specific residues to Rot function. Rot was found to form a dimer, with each monomer harboring a winged helix-turn-helix (WHTH) DNA-binding motif. Despite an overall acidic pI, the asymmetric electrostatic charge profile suggests that Rot can orient the WHTH domain to bind DNA. Structure-based site-directed mutagenesis studies demonstrated that 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. As a result, this work provides insight into a precise mechanism by which Rot controls virulence factor regulation in S. aureus.« less

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

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

Killikelly, April; Jakoncic, Jean; Benson, Meredith A.

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 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. As a result, this work provides insight into a precise mechanism by which Rot controls virulence factor regulation in S. aureus.« less

DND protein functions as a translation repressor during zebrafish embryogenesis.

PubMed

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

2017-03-04

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

dREAM co-operates with insulator-binding proteins and regulates expression at divergently paired genes

PubMed Central

Korenjak, Michael; Kwon, Eunjeong; Morris, Robert T.; Anderssen, Endre; Amzallag, Arnaud; Ramaswamy, Sridhar; Dyson, Nicholas J.

2014-01-01

dREAM complexes represent the predominant form of E2F/RBF repressor complexes in Drosophila. dREAM associates with thousands of sites in the fly genome but its mechanism of action is unknown. To understand the genomic context in which dREAM acts we examined the distribution and localization of Drosophila E2F and dREAM proteins. Here we report a striking and unexpected overlap between dE2F2/dREAM sites and binding sites for the insulator-binding proteins CP190 and Beaf-32. Genetic assays show that these components functionally co-operate and chromatin immunoprecipitation experiments on mutant animals demonstrate that dE2F2 is important for association of CP190 with chromatin. dE2F2/dREAM binding sites are enriched at divergently transcribed genes, and the majority of genes upregulated by dE2F2 depletion represent the repressed half of a differentially expressed, divergently transcribed pair of genes. Analysis of mutant animals confirms that dREAM and CP190 are similarly required for transcriptional integrity at these gene pairs and suggest that dREAM functions in concert with CP190 to establish boundaries between repressed/activated genes. Consistent with the idea that dREAM co-operates with insulator-binding proteins, genomic regions bound by dREAM possess enhancer-blocking activity that depends on multiple dREAM components. These findings suggest that dREAM functions in the organization of transcriptional domains. PMID:25053843

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

PubMed Central

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

2002-01-01

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

Fli1 Represses Transcription of the Human α2(I) Collagen Gene by Recruitment of the HDAC1/p300 Complex

PubMed Central

Asano, Yoshihide; Trojanowska, Maria

2013-01-01

Fli1, a member of the Ets transcription factor family, is a key repressor of the human α2(I) collagen (COL1A2) gene. Although our previous studies have delineated that TGF-β induces displacement of Fli1 from the COL1A2 promoter through sequential post-translational modifications, the detailed mechanism by which Fli1 functions as a potent transcriptional repressor of the COL1A2 gene has not been fully investigated. To address this issue, we carried out a series of experiments especially focusing on protein-protein interaction and epigenetic transcriptional regulation. The combination of tandem affinity purification and mass spectrometry identified HDAC1 as a Fli1 interacting protein. Under quiescent conditions, HDAC1 induced deacetylation of Fli1 resulting in an increase of Fli1 DNA binding ability and p300 enhanced this process by promoting the formation of a Fli1-HDAC1-p300 complex. TGF-β-induced phosphorylation of Fli1 at threonine 312 led to disassembly of this protein complex. In quiescent dermal fibroblasts Fli1, HDAC1, and p300 occupied the −404 to −237 region, including the Fli1 binding site, of the COL1A2 promoter. TGF-β induced Fli1 and HDAC1 dissociation from the COL1A2 promoter, while promoting Ets1 and p300 recruitment. Furthermore, acetylation levels of histone H3 around the Fli1 binding site in the COL1A2 promoter inversely correlated with the DNA occupancy of Fli1 and HDAC1, while positively correlating with that of Ets1 and p300. In the functional studies, HDAC1 overexpression magnified the inhibitory effect of Fli1 on the COL1A2 promoter. Moreover, pharmacological blockade of HDAC1 by entinostat enhanced collagen production in dermal fibroblasts. Collectively, these results indicate that under quiescent conditions Fli1 recruits HDAC1/p300 to the COL1A2 promoter and suppresses the expression of the COL1A2 gene by chromatin remodeling through histone deacetylation. TGF-β-dependent phosphorylation of Fli1 at threonine 312 is a critical step regulating the remodeling of the Fli1 transcription repressor complex, leading to transcriptional activation of the COL1A2 gene. PMID:24058639

Cdyl: a new transcriptional co-repressor

PubMed Central

Caron, Cécile; Pivot-Pajot, Christophe; van Grunsven, Leo A.; Col, Edwige; Lestrat, Cécile; Rousseaux, Sophie; Khochbin, Saadi

2003-01-01

Cdyl (chromodomain-Y-like) is a chromodomain-containing protein that is predominantly expressed during mouse spermiogenesis. In its carboxy-terminal portion, there is a domain with homology to the coenzyme A (CoA) pocket of the enoyl-CoA hydratase/isomerase, which is shown here to be able to bind CoA and histone deacetylases (HDACs). It also efficiently represses transcription. Moreover, the binding of Hdac1 represses the ability of Cdyl to bind CoA, and a Cdyl–CoA interaction only occurs in the absence of HDACs. These data suggest that Cdyl is primarily a transcriptional co-repressor. However, the degradation of cellular Hdac1 and Hdac2, as observed here in the elongating spermatids, may provide an HDAC-free environment in which Cdyl could bind CoA and participate in the global chromatin remodelling that occurs in these cells. PMID:12947414

Comprehensive Interrogation of Natural TALE DNA Binding Modules and Transcriptional Repressor Domains

PubMed Central

Cong, Le; Zhou, Ruhong; Kuo, Yu-chi; Cunniff, Margaret; Zhang, Feng

2012-01-01

Transcription activator-like effectors (TALE) are sequence-specific DNA binding proteins that harbor modular, repetitive DNA binding domains. TALEs have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the TALE toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat variable diresidue (RVD) Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective TALE transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using TALEs. PMID:22828628

Bacillus subtilis IolQ (DegA) is a transcriptional repressor of iolX encoding NAD+-dependent scyllo-inositol dehydrogenase.

PubMed

Kang, Dong-Min; Michon, Christophe; Morinaga, Tetsuro; Tanaka, Kosei; Takenaka, Shinji; Ishikawa, Shu; Yoshida, Ken-Ichi

2017-07-11

Bacillus subtilis is able to utilize at least three inositol stereoisomers as carbon sources, myo-, scyllo-, and D-chiro-inositol (MI, SI, and DCI, respectively). NAD + -dependent SI dehydrogenase responsible for SI catabolism is encoded by iolX. Even in the absence of functional iolX, the presence of SI or MI in the growth medium was found to induce the transcription of iolX through an unknown mechanism. Immediately upstream of iolX, there is an operon that encodes two genes, yisR and iolQ (formerly known as degA), each of which could encode a transcriptional regulator. Here we performed an inactivation analysis of yisR and iolQ and found that iolQ encodes a repressor of the iolX transcription. The coding sequence of iolQ was expressed in Escherichia coli and the gene product was purified as a His-tagged fusion protein, which bound to two sites within the iolX promoter region in vitro. IolQ is a transcriptional repressor of iolX. Genetic evidences allowed us to speculate that SI and MI might possibly be the intracellular inducers, however they failed to antagonize DNA binding of IolQ in in vitro experiments.

Covalent attachment of Arc repressor subunits by a peptide linker enhances affinity for operator DNA.

PubMed

Robinson, C R; Sauer, R T

1996-01-09

By designing a recombinant gene containing tandem copies of the arc coding sequence with intervening DNA encoding the linker sequence GGGSGGGTGGGSGGG, the two subunits of the P22 Are repressor dimer have been covalently linked to form a single-chain protein called Arc-L1-Arc. The 15-residue linker joins the C-terminus of one monomer to the N-terminus of the second, a distance of approximately 45 A in the Arc-operator cocrystal structure. Arc-L1-Arc is expressed at high levels in Escherichia coli, with no evidence of degradation or proteolytic clipping of the linker, and is more active than wild-type Arc in repression assays. The purified Arc-L1-Arc protein has the molecular weight expected for the designed protein and unfolds cooperatively, reversibly, and with no concentration dependence in thermal-denaturation studies. Arc-L1-Arc protects operator DNA in a manner indistinguishable from that of wild-type Arc in DNase I and copper-phenanthroline footprinting studies, but the covalent attachment of the two monomers results in enhanced affinity for operator DNA. Arc-L1-Arc binds operator DNA half-maximally at a concentration of 1.7 pM, compared with the wild-type value of 185 pM, and also binds DNA fragments containing the left or right operator half-sites more tightly than wild type. Because wild-type Arc is monomeric at sub-nanomolar concentrations and must dimerize before binding to the operator, it was anticipated that Arc-L1-Arc would exhibit a lower half-maximal binding concentration. However, even when the change from a monomeric to a dimeric species is taken into account, the affinity of Arc-L1-Arc for operator and half-operator DNA is greater than the wild-type affinity. This tighter binding appears to result from slower dissociation, as Arc-L1-Arc DNA complexes with full or half-site operators dissociate at rates 5-10 times slower than the corresponding Arc--DNA complexes. Hence, the activity of the designed Arc-L1-Arc protein is substantially increased relative to wild-type Arc in a variety of assays.

Structural and Functional Analysis of the Human HDAC4 Catalytic Domain Reveals a Regulatory Structural Zinc-binding Domain*S⃞

PubMed Central

Bottomley, Matthew J.; Lo Surdo, Paola; Di Giovine, Paolo; Cirillo, Agostino; Scarpelli, Rita; Ferrigno, Federica; Jones, Philip; Neddermann, Petra; De Francesco, Raffaele; Steinkühler, Christian; Gallinari, Paola; Carfí, Andrea

2008-01-01

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR·HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions. PMID:18614528

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

PubMed

Bottomley, Matthew J; Lo Surdo, Paola; Di Giovine, Paolo; Cirillo, Agostino; Scarpelli, Rita; Ferrigno, Federica; Jones, Philip; Neddermann, Petra; De Francesco, Raffaele; Steinkühler, Christian; Gallinari, Paola; Carfí, Andrea

2008-09-26

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.

Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in l-lactate and sugar utilization

PubMed Central

Gao, Yong-Gui; Suzuki, Hiroaki; Itou, Hiroshi; Zhou, Yong; Tanaka, Yoshikazu; Wachi, Masaaki; Watanabe, Nobuhisa; Tanaka, Isao; Yao, Min

2008-01-01

LldR (CGL2915) from Corynebacterium glutamicum is a transcription factor belonging to the GntR family, which is typically involved in the regulation of oxidized substrates associated with amino acid metabolism. In the present study, the crystal structure of LldR was determined at 2.05-Å resolution. The structure consists of N- and C-domains similar to those of FadR, but with distinct domain orientations. LldR and FadR dimers achieve similar structures by domain swapping, which was first observed in dimeric assembly of transcription factors. A structural feature of Zn2+ binding in the regulatory domain was also observed, as a difference from the FadR subfamily. DNA microarray and DNase I footprint analyses suggested that LldR acts as a repressor regulating cgl2917-lldD and cgl1934-fruK-ptsF operons, which are indispensable for l-lactate and fructose/sucrose utilization, respectively. Furthermore, the stoichiometries and affinities of LldR and DNAs were determined by isothermal titration calorimetry measurements. The transcriptional start site and repression of LldR on the cgl2917-lldD operon were analysed by primer extension assay. Mutation experiments showed that residues Lys4, Arg32, Arg42 and Gly63 are crucial for DNA binding. The location of the putative ligand binding cavity and the regulatory mechanism of LldR on its affinity for DNA were proposed. PMID:18988622

Leveraging cross-species transcription factor binding site patterns: from diabetes risk loci to disease mechanisms.

PubMed

Claussnitzer, Melina; Dankel, Simon N; Klocke, Bernward; Grallert, Harald; Glunk, Viktoria; Berulava, Tea; Lee, Heekyoung; Oskolkov, Nikolay; Fadista, Joao; Ehlers, Kerstin; Wahl, Simone; Hoffmann, Christoph; Qian, Kun; Rönn, Tina; Riess, Helene; Müller-Nurasyid, Martina; Bretschneider, Nancy; Schroeder, Timm; Skurk, Thomas; Horsthemke, Bernhard; Spieler, Derek; Klingenspor, Martin; Seifert, Martin; Kern, Michael J; Mejhert, Niklas; Dahlman, Ingrid; Hansson, Ola; Hauck, Stefanie M; Blüher, Matthias; Arner, Peter; Groop, Leif; Illig, Thomas; Suhre, Karsten; Hsu, Yi-Hsiang; Mellgren, Gunnar; Hauner, Hans; Laumen, Helmut

2014-01-16

Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2 diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression

PubMed Central

Abruzzi, Katharine Compton; Rodriguez, Joseph; Menet, Jerome S.; Desrochers, Jennifer; Zadina, Abigail; Luo, Weifei; Tkachev, Sasha; Rosbash, Michael

2011-01-01

CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation (ChIP) tiling array assays (ChIP–chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4–6 h later by the transcriptional repressor PERIOD (PER), indicating that the majority of CLK targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation. PMID:22085964

Operator design and mechanism for CarA repressor-mediated down-regulation of the photoinducible carB operon in Myxococcus xanthus.

PubMed

López-Rubio, José Juan; Padmanabhan, S; Lázaro, Jose María; Salas, Margarita; Murillo, Francisco José; Elías-Arnanz, Montserrat

2004-07-09

The carB operon encodes all except one of the enzymes involved in light-induced carotenogenesis in Myxococcus xanthus. Expression of its promoter (P(B)) is repressed in the dark by sequence-specific DNA binding of CarA to a palindrome (pI) located between positions -47 and -64 relative to the transcription start site. This promotes subsequent binding of CarA to additional sites that remain to be defined. CarS, produced in the light, interacts physically with CarA, abrogates CarA-DNA binding, and thereby derepresses P(B). In this study, we delineate the operator design that exists for CarA by precisely mapping out the second operator element. For this, we examined how stepwise deletions and site-directed mutagenesis in the region between the palindrome and the transcription start site affect CarA binding around P(B) in vitro and expression of P(B) in vivo. These revealed the second operator element to be an imperfect interrupted palindrome (pII) spanning positions -26 to -40. In vitro assays using purified M. xanthus RNA polymerase showed that CarA abolishes P(B)-RNA polymerase binding and runoff transcription and that both were restored by CarS, thus rationalizing the observations in vivo. CarA binding to pII (after association with pI) effectively occludes RNA polymerase from P(B) and so provides the operative mechanism for the repression of the carB operon by CarA. The bipartite operator design, whereby transcription is blocked by the low affinity CarA-pII binding and is readily restored by CarS, may have evolved to match the needs for a rapid and an effective response to light.

Functional diversification of ROK-family transcriptional regulators of sugar catabolism in the Thermotogae phylum

PubMed Central

Kazanov, Marat D.; Li, Xiaoqing; Gelfand, Mikhail S.; Osterman, Andrei L.; Rodionov, Dmitry A.

2013-01-01

Large and functionally heterogeneous families of transcription factors have complex evolutionary histories. What shapes specificities toward effectors and DNA sites in paralogous regulators is a fundamental question in biology. Bacteria from the deep-branching lineage Thermotogae possess multiple paralogs of the repressor, open reading frame, kinase (ROK) family regulators that are characterized by carbohydrate-sensing domains shared with sugar kinases. We applied an integrated genomic approach to study functions and specificities of regulators from this family. A comparative analysis of 11 Thermotogae genomes revealed novel mechanisms of transcriptional regulation of the sugar utilization networks, DNA-binding motifs and specific functions. Reconstructed regulons for seven groups of ROK regulators were validated by DNA-binding assays using purified recombinant proteins from the model bacterium Thermotoga maritima. All tested regulators demonstrated specific binding to their predicted cognate DNA sites, and this binding was inhibited by specific effectors, mono- or disaccharides from their respective sugar catabolic pathways. By comparing ligand-binding domains of regulators with structurally characterized kinases from the ROK family, we elucidated signature amino acid residues determining sugar-ligand regulator specificity. Observed correlations between signature residues and the sugar-ligand specificities provide the framework for structure functional classification of the entire ROK family. PMID:23209028

Cellular homeoproteins, SATB1 and CDP, bind to the unique region between the human cytomegalovirus UL127 and major immediate-early genes

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

Lee Jialing; Klase, Zachary; Gao Xiaoqi

An AT-rich region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region (UR). It has been shown that the UR represses activation of transcription from the UL127 promoter and functions as a boundary between the divergent UL127 and MIE genes during human CMV infection [Angulo, A., Kerry, D., Huang, H., Borst, E.M., Razinsky, A., Wu, J., Hobom, U., Messerle, M., Ghazal, P., 2000. Identification of a boundary domain adjacent to the potent human cytomegalovirus enhancer that represses transcription of the divergent UL127 promoter. J.more » Virol. 74 (6), 2826-2839; Lundquist, C.A., Meier, J.L., Stinski, M.F., 1999. A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer. J. Virol. 73 (11), 9039-9052]. A putative forkhead box-like (FOX-like) site, AAATCAATATT, was identified in the UR and found to play a key role in repression of the UL127 promoter in recombinant virus-infected cells [Lashmit, P.E., Lundquist, C.A., Meier, J.L., Stinski, M.F., 2004. Cellular repressor inhibits human cytomegalovirus transcription from the UL127 promoter. J. Virol. 78 (10), 5113-5123]. However, the cellular factors which associate with the UR and FOX-like region remain to be determined. We reported previously that pancreatic-duodenal homeobox factor-1 (PDX1) bound to a 45-bp element located within the UR [Chao, S.H., Harada, J.N., Hyndman, F., Gao, X., Nelson, C.G., Chanda, S.K., Caldwell, J.S., 2004. PDX1, a Cellular Homeoprotein, Binds to and Regulates the Activity of Human Cytomegalovirus Immediate Early Promoter. J. Biol. Chem. 279 (16), 16111-16120]. Here we demonstrate that two additional cellular homeoproteins, special AT-rich sequence binding protein 1 (SATB1) and CCAAT displacement protein (CDP), bind to the human CMV UR in vitro and in vivo. Furthermore, CDP is identified as a FOX-like binding protein and a repressor of the UL127 promoter, while SATB1 has no effect on UL127 expression. Since CDP is known as a transcription repressor and a nuclear matrix-associated region binding protein, CDP may have a role in the regulation of human CMV transcription.« less

Light-induced carotenogenesis in Myxococcus xanthus: evidence that CarS acts as an anti-repressor of CarA.

PubMed

Whitworth, D E; Hodgson, D A

2001-11-01

In the bacterium Myxococcus xanthus, carotenoids are produced in response to illumination, as a result of expression of the crt carotenoid biosynthesis genes. The majority of crt genes are clustered in the crtEBDC operon, which is repressed in the dark by CarA. Genetic data suggest that, in the light, CarS is synthesized and achieves activation of the crtEBDC operon by removing the repressive action of CarA. As CarS contains no known DNA-binding motif, the relief of CarA-mediated repression was postulated to result from a direct interaction between these two proteins. Use of the yeast two-hybrid system demonstrated direct interaction between CarA and CarS. The two-hybrid system also implied that CarA and, possibly, CarS are capable of homodimerization. Direct evidence for CarS anti-repressor action was provided in vitro. A glutathione S-transferase (GST)-CarA protein fusion was shown to bind specifically to a palindromic operator sequence within the crtEBDC promoter. CarA was prevented from binding to its operator, and prebound CarA was removed by the addition of purified CarS. CarS is therefore an anti-repressor.

Design, Synthesis, and Evaluation of Novel p-(methylthio)styryl Substituted Quindoline Derivatives as Neuroblastoma RAS (NRAS) Repressors via Specific Stabilizing the RNA G-Quadruplex.

PubMed

Peng, Wang; Sun, Zhi-Yin; Zhang, Qi; Cheng, Sui-Qi; Wang, Shi-Ke; Wang, Xiao-Na; Kuang, Guo-Tao; Su, Xiao-Xuan; Tan, Jia-Heng; Huang, Zhi-Shu; Ou, Tian-Miao

2018-05-25

The human proto-oncogene neuroblastoma RAS (NRAS) contains a guanine-rich sequence in the 5'-untranslated regions (5'-UTR) of the mRNA that could form an RNA G-quadruplex structure. This structure acts as a repressor for NRAS translation and could be a potential target for anti-cancer drugs. Our previous studies found an effective scaffold, the quindoline scaffold, for binding and stabilizing the DNA G-quadruplex structures. Here, basing on the previous studies and reported RNA-specific probes, a series of novel p-(methylthio)styryl substituted quindoline (MSQ) derivatives were designed, synthesized and evaluated as NRAS RNA G-quadruplex ligands. Panels of experiments turned out that the introduction of p-(methylthio)styryl side chain could enhance the specific binding to the NRAS RNA G-quadruplex. One of the hits, 4a-10, showed strong stabilizing activity on the G-quadruplex, and subsequently repressed NRAS's translation and inhibited tumor cells proliferation. Our finding provided a novel strategy to discover novel NRAS repressors by specifically binding to the RNA G-quadruplex in the 5'-UTR of mRNA.

Light-dependent gene regulation by a coenzyme B12-based photoreceptor

PubMed Central

Ortiz-Guerrero, Juan Manuel; Polanco, María Carmen; Murillo, Francisco J.; Padmanabhan, S.; Elías-Arnanz, Montserrat

2011-01-01

Cobalamin (B12) typically functions as an enzyme cofactor but can also regulate gene expression via RNA-based riboswitches. B12-directed gene regulatory mechanisms via protein factors have, however, remained elusive. Recently, we reported down-regulation of a light-inducible promoter in the bacterium Myxococcus xanthus by two paralogous transcriptional repressors, of which one, CarH, but not the other, CarA, absolutely requires B12 for activity even though both have a canonical B12-binding motif. Unanswered were what underlies this striking difference, what is the specific cobalamin used, and how it acts. Here, we show that coenzyme B12 (5′-deoxyadenosylcobalamin, AdoB12), specifically dictates CarH function in the dark and on exposure to light. In the dark, AdoB12-binding to the autonomous domain containing the B12-binding motif foments repressor oligomerization, enhances operator binding, and blocks transcription. Light, at various wavelengths at which AdoB12 absorbs, dismantles active repressor oligomers by photolysing the bound AdoB12 and weakens repressor–operator binding to allow transcription. By contrast, AdoB12 alters neither CarA oligomerization nor operator binding, thus accounting for its B12-independent activity. Our findings unveil a functional facet of AdoB12 whereby it serves as the chromophore of a unique photoreceptor protein class acting in light-dependent gene regulation. The prevalence of similar proteins of unknown function in microbial genomes suggests that this distinct B12-based molecular mechanism for photoregulation may be widespread in bacteria. PMID:21502508

Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor.

PubMed

Burkart, Anna D; Mukherjee, Abir; Mayo, Kelly E

2006-03-01

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

Nitric oxide sensitizes tumor cells to TRAIL-induced apoptosis via inhibition of the DR5 transcription repressor Yin Yang 1.

PubMed

Huerta-Yepez, Sara; Vega, Mario; Escoto-Chavez, Saul E; Murdock, Benjamin; Sakai, Toshiyuki; Baritaki, Stavroula; Bonavida, Benjamin

2009-02-01

Treatment of TRAIL-resistant tumor cells with the nitric oxide donor DETANONOate sensitizes the tumor cells to TRAIL-induced apoptosis concomitantly with DR5 upregulation. The mechanism of sensitization was examined based on the hypothesis that DETANONOate inhibits a transcription repressor Yin Yang 1 (YY1) that negatively regulates DR5 transcription. Treatment of the prostate carcinoma cell lines with DETANONOate inhibited both NF-kappaB and YY1 DNA-binding activities concomitantly with upregulation of DR5 expression. The direct role of YY1 in the regulation of TRAIL resistance was demonstrated in cells treated with YY1 siRNA resulting in TRAIL-induced apoptosis. The role of YY1 in the transcriptional regulation of DR5 was examined in cells treated with a DR5 luciferase reporter system (pDR5) and two constructs, namely, the pDR5/-605 construct with a deletion of the putative YY1 DNA-binding region (-1224 to -605) and a construct pDR5-YY1 with a mutation of the YY1 DNA-binding site. A significant (3-fold) augmentation of luciferase activity over baseline transfection with pDR5 was observed in cells transfected with the modified constructs. ChIP analysis corroborated the YY1 binding to the DR5 promoter. In vivo, tissues from nude mice bearing the PC-3 xenograft and treated with DETANONOate showed inhibition of YY1 and upregulation of DR5. The present findings demonstrate that YY1 negatively regulates DR5 transcription and expression and these correlated with resistance to TRAIL-induced apoptosis. DETANONOate inhibits both NF-kappaB and YY1 and in combination with TRAIL reverses tumor cell resistance to TRAIL apoptosis.

Genetic control of osmoadaptive glycine betaine synthesis in Bacillus subtilis through the choline-sensing and glycine betaine-responsive GbsR repressor.

PubMed

Nau-Wagner, Gabriele; Opper, Daniela; Rolbetzki, Anne; Boch, Jens; Kempf, Bettina; Hoffmann, Tamara; Bremer, Erhard

2012-05-01

Synthesis of the compatible solute glycine betaine confers a considerable degree of osmotic stress tolerance to Bacillus subtilis. This osmoprotectant is produced through the uptake of the precursor choline via the osmotically inducible OpuB and OpuC ABC transporters and a subsequent two-step oxidation process by the GbsB and GbsA enzymes. We characterized a regulatory protein, GbsR, controlling the transcription of both the structural genes for the glycine betaine biosynthetic enzymes (gbsAB) and those for the choline-specific OpuB transporter (opuB) but not of that for the promiscuous OpuC transporter. GbsR acts genetically as a repressor and functions as an intracellular choline sensor. Spectroscopic analysis of the purified GbsR protein showed that it binds the inducer choline with an apparent K(D) (equilibrium dissociation constant) of approximately 165 μM. Based on the X-ray structure of a protein (Mj223) from Methanococcus jannaschii, a homology model for GbsR was derived. Inspection of this GbsR in silico model revealed a possible ligand-binding pocket for choline resembling those of known choline-binding sites present in solute receptors of microbial ABC transporters, e.g., that of the OpuBC ligand-binding protein of the OpuB ABC transporter. GbsR was not only needed to control gbsAB and opuB expression in response to choline availability but also required to genetically tune down glycine betaine production once cellular adjustment to high osmolarity has been achieved. The GbsR regulatory protein from B. subtilis thus records and integrates cellular and environmental signals for both the onset and the repression of the synthesis of the osmoprotectant glycine betaine.

Genetic Control of Osmoadaptive Glycine Betaine Synthesis in Bacillus subtilis through the Choline-Sensing and Glycine Betaine-Responsive GbsR Repressor

PubMed Central

Nau-Wagner, Gabriele; Opper, Daniela; Rolbetzki, Anne; Boch, Jens; Kempf, Bettina; Hoffmann, Tamara

2012-01-01

Synthesis of the compatible solute glycine betaine confers a considerable degree of osmotic stress tolerance to Bacillus subtilis. This osmoprotectant is produced through the uptake of the precursor choline via the osmotically inducible OpuB and OpuC ABC transporters and a subsequent two-step oxidation process by the GbsB and GbsA enzymes. We characterized a regulatory protein, GbsR, controlling the transcription of both the structural genes for the glycine betaine biosynthetic enzymes (gbsAB) and those for the choline-specific OpuB transporter (opuB) but not of that for the promiscuous OpuC transporter. GbsR acts genetically as a repressor and functions as an intracellular choline sensor. Spectroscopic analysis of the purified GbsR protein showed that it binds the inducer choline with an apparent KD (equilibrium dissociation constant) of approximately 165 μM. Based on the X-ray structure of a protein (Mj223) from Methanococcus jannaschii, a homology model for GbsR was derived. Inspection of this GbsR in silico model revealed a possible ligand-binding pocket for choline resembling those of known choline-binding sites present in solute receptors of microbial ABC transporters, e.g., that of the OpuBC ligand-binding protein of the OpuB ABC transporter. GbsR was not only needed to control gbsAB and opuB expression in response to choline availability but also required to genetically tune down glycine betaine production once cellular adjustment to high osmolarity has been achieved. The GbsR regulatory protein from B. subtilis thus records and integrates cellular and environmental signals for both the onset and the repression of the synthesis of the osmoprotectant glycine betaine. PMID:22408163

Regulation and Adaptive Evolution of Lactose Operon Expression in Lactobacillus delbrueckii

PubMed Central

Lapierre, Luciane; Mollet, Beat; Germond, Jacques-Edouard

2002-01-01

Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis are both used in the dairy industry as homofermentative lactic acid bacteria in the production of fermented milk products. After selective pressure for the fast fermentation of milk in the manufacture of yogurts, L. delbrueckii subsp. bulgaricus loses its ability to regulate lac operon expression. A series of mutations led to the constitutive expression of the lac genes. A complex of insertion sequence (IS) elements (ISL4 inside ISL5), inserted at the border of the lac promoter, induced the loss of the palindromic structure of one of the operators likely involved in the binding of regulatory factors. A lac repressor gene was discovered downstream of the β-galactosidase gene of L. delbrueckii subsp. lactis and was shown to be inactivated by several mutations in L. delbrueckii subsp. bulgaricus. Regulatory mechanisms of the lac gene expression of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis were compared by heterologous expression in Lactococcus lactis of the two lac promoters in front of a reporter gene (β-glucuronidase) in the presence or absence of the lac repressor gene. Insertion of the complex of IS elements in the lac promoter of L. delbrueckii subsp. bulgaricus increased the promoter's activity but did not prevent repressor binding; rather, it increased the affinity of the repressor for the promoter. Inactivation of the lac repressor by mutations was then necessary to induce the constitutive expression of the lac genes in L. delbrueckii subsp. bulgaricus. PMID:11807052

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

PubMed Central

Ishiai, M; Wada, C; Kawasaki, Y; Yura, T

1994-01-01

Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication. Images PMID:8170998

The Arabidopsis class I TCP transcription factor AtTCP11 is a developmental regulator with distinct DNA-binding properties due to the presence of a threonine residue at position 15 of the TCP domain.

PubMed

Viola, Ivana L; Uberti Manassero, Nora G; Ripoll, Rodrigo; Gonzalez, Daniel H

2011-04-01

The TCP domain is a DNA-binding domain present in plant transcription factors that modulate different processes. In the present study, we show that Arabidopsis class I TCP proteins are able to interact with a dyad-symmetric sequence composed of two GTGGG half-sites. TCP20 establishes symmetric interactions with the 5' half of each strand, whereas TCP11 interacts mainly with the 3' half. SELEX (systematic evolution of ligands by exponential enrichment) experiments with TCP15 and TCP20 indicated that these proteins have similar, although not identical, DNA-binding preferences and are able to interact with non-palindromic binding sites of the type GTGGGNCCNN. TCP11 shows a different DNA-binding specificity, with a preference for the sequence GTGGGCCNNN. The distinct DNA-binding properties of TCP11 are due to the presence of a threonine residue at position 15 of the TCP domain, a position that is occupied by an arginine residue in most TCP proteins. TCP11 also forms heterodimers with TCP15 that have increased DNA-binding efficiency. The expression in plants of a repressor form of TCP11 demonstrated that this protein is a developmental regulator that influences the growth of leaves, stems and petioles, and pollen development. The results suggest that changes in DNA-binding preferences may be one of the mechanisms through which class I TCP proteins achieve functional specificity.

Deciphering the combinatorial architecture of a Drosophila homeotic gene enhancer

PubMed Central

Drewell, Robert A.; Nevarez, Michael J.; Kurata, Jessica S.; Winkler, Lauren N.; Li, Lily; Dresch, Jacqueline M.

2013-01-01

Summary In Drosophila, the 330 kb bithorax complex regulates cellular differentiation along the anterio-posterior axis during development in the thorax and abdomen and is comprised of three homeotic genes: Ultrabithorax, abdominal-A, and Abdominal-B. The expression of each of these genes is in turn controlled through interactions between transcription factors and a number of cis-regulatory modules in the neighboring intergenic regions. In this study, we examine how the sequence architecture of transcription factor binding sites mediates the functional activity of one of these cis-regulatory modules. Using computational, mathematical modeling and experimental molecular genetic approaches we investigate the IAB7b enhancer, which regulates Abdominal-B expression specifically in the presumptive seventh and ninth abdominal segments of the early embryo. A cross-species comparison of the IAB7b enhancer reveals an evolutionarily conserved signature motif containing two FUSHI-TARAZU activator transcription factor binding sites. We find that the transcriptional repressors KNIRPS, KRUPPEL and GIANT are able to restrict reporter gene expression to the posterior abdominal segments, using different molecular mechanisms including short-range repression and competitive binding. Additionally, we show the functional importance of the spacing between the two FUSHI-TARAZU binding sites and discuss the potential importance of cooperativity for transcriptional activation. Our results demonstrate that the transcriptional output of the IAB7b cis-regulatory module relies on a complex set of combinatorial inputs mediated by specific transcription factor binding and that the sequence architecture at this enhancer is critical to maintain robust regulatory function. PMID:24514265

Characterization of the colorectal cancer-associated enhancer MYC-335 at 8q24: the role of rs67491583.

PubMed

Tuupanen, Sari; Yan, Jian; Turunen, Mikko; Gylfe, Alexandra E; Kaasinen, Eevi; Li, Li; Eng, Charis; Culver, Daniel A; Kalady, Matthew F; Pennison, Michael J; Pasche, Boris; Manne, Upender; de la Chapelle, Albert; Hampel, Heather; Henderson, Brian E; Marchand, Loic Le; Hautaniemi, Sampsa; Askhtorab, Hassan; Smoot, Duane; Sandler, Robert S; Keku, Temitope; Kupfer, Sonia S; Ellis, Nathan A; Haiman, Christopher A; Taipale, Jussi; Aaltonen, Lauri A

2012-01-01

Recent genome-wide association studies have identified multiple regions at 8q24 that confer susceptibility to many cancers. In our previous work, we showed that the colorectal cancer (CRC) risk variant rs6983267 at 8q24 resides within a TCF4 binding site at the MYC-335 enhancer, with the risk allele G having a stronger binding capacity and Wnt responsiveness. Here, we searched for other potential functional variants within MYC-335. Genetic variation within MYC-335 was determined in samples from individuals of European, African, and Asian descent, with emphasis on variants in putative transcription factor binding sites. A 2-bp GA deletion rs67491583 was found to affect a growth factor independent (GFI) binding site and was present only in individuals with African ancestry. Chromatin immunoprecipitation performed in heterozygous cells showed that the GA deletion had an ability to reduce binding of the transcriptional repressors GFI1 and GFI1b. Screening of 1,027 African American colorectal cancer cases and 1,773 healthy controls did not reveal evidence for association (odds ratio: 1.17, 95% confidence interval: 0.97-1.41, P = 0.095). In this study, rs67491583 was identified as another functional variant in the CRC-associated enhancer MYC-335, but further studies are needed to establish the role of rs67491583 in the colorectal cancer predisposition of African Americans. Copyright © 2012 Elsevier Inc. All rights reserved.

Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator

PubMed Central

Rajasekar, Karthik V.; Lovering, Andrew L.; Dancea, Felician; Scott, David J.; Harris, Sarah A.; Bingle, Lewis E.H.; Roessle, Manfred; Thomas, Christopher M.; Hyde, Eva I.; White, Scott A.

2016-01-01

Abstract The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53. PMID:27016739

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

PubMed

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

2009-08-01

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

Suppressor of Fused Chaperones Gli Proteins To Generate Transcriptional Responses to Sonic Hedgehog Signaling

PubMed Central

Zhang, Ziyu; Shen, Longyan; Law, Kelvin; Zhang, Zengdi; Liu, Xiaotong; Hua, Hu; Li, Sanen; Huang, Huijie; Yue, Shen; Hui, Chi-chung

2016-01-01

ABSTRACT Cellular responses to the graded Sonic Hedgehog (Shh) morphogenic signal are orchestrated by three Gli genes that give rise to both transcription activators and repressors. An essential downstream regulator of the pathway, encoded by the tumor suppressor gene Suppressor of fused (Sufu), plays critical roles in the production, trafficking, and function of Gli proteins, but the mechanism remains controversial. Here, we show that Sufu is upregulated in active Shh responding tissues and accompanies Gli activators translocating into and Gli repressors out of the nucleus. Trafficking of Sufu to the primary cilium, potentiated by Gli activators but not repressors, was found to be coupled to its nuclear import. We have identified a nuclear export signal (NES) motif of Sufu in juxtaposition to the protein kinase A (PKA) and glycogen synthase kinase 3 (GSK3) dual phosphorylation sites and show that Sufu binds the chromatin with both Gli1 and Gli3. Close comparison of neural tube development among individual Ptch1−/−, Sufu−/−, and Ptch1−/−; Sufu−/− double mutant embryos indicates that Sufu is critical for the maximal activation of Shh signaling essential to the specification of the most-ventral neurons. These data define Sufu as a novel class of molecular chaperone required for every aspect of Gli regulation and function. PMID:27849569

Autoregulation and Virulence Control by the Toxin-Antitoxin System SavRS in Staphylococcus aureus

PubMed Central

Wen, Wen; Liu, Banghui; Xue, Lu; Zhu, Zhongliang; Niu, Liwen

2018-01-01

ABSTRACT Toxin-antitoxin (TA) systems play diverse physiological roles, such as plasmid maintenance, growth control, and persister cell formation, but their involvement in bacterial pathogenicity remains largely unknown. Here, we have identified a novel type II toxin-antitoxin system, SavRS, and revealed the molecular mechanisms of its autoregulation and virulence control in Staphylococcus aureus. Electrophoretic mobility shift assay and isothermal titration calorimetry data indicated that the antitoxin SavR acted as the primary repressor bound to its own promoter, while the toxin SavS formed a complex with SavR to enhance the ability to bind to the operator site. DNase I footprinting assay identified the SavRS-binding site containing a short and long palindrome in the promoter region. Further, mutation and DNase I footprinting assay demonstrated that the two palindromes were crucial for DNA binding and transcriptional repression. More interestingly, genetic deletion of the savRS system led to the increased hemolytic activity and pathogenicity in a mouse subcutaneous abscess model. We further identified two virulence genes, hla and efb, by real-time quantitative reverse transcription-PCR and demonstrated that SavR and SavRS could directly bind to their promoter regions to repress virulence gene expression. PMID:29440365

tif-1 mutation alters polynucleotide recognition by the recA protein of Escherichia coli.

PubMed Central

McEntee, K; Weinstock, G M

1981-01-01

The requirements for polynucleotide-dependent hydrolysis of ATP and for proteolytic cleavage of phage lambda repressor have been examined for both the wild-type (recA+ protein) and the tif-1 mutant form [tif(recA) protein] of the recA gene product. The recA+ and tif(recA) proteins catalyze both reactions in the presence of long single-stranded DNAs or certain deoxyhomopolymers. However, short oligonucleotides [(dT)12, (dA)14] stimulate neither the protease nor the ATPase activities of the recA+ protein. In contrast, these short oligonucleotides activate tif(recA) protein to cleave lambda repressor without stimulating its ATPase activity. Moreover, both the ATPase and protease activities of the tif(recA) protein are stimulated by poly(rU) and poly(rC) whereas the recA+ protein does not respond to these ribopolymers. We have purified the recA protein from a strain in which the tif mutation is intragenically suppressed. This mutant protein (recA629) is inactive in the presence of (dT)12, (dA)14, poly(rU), and poly(rC) for lambda repressor cleavage and ATP hydrolysis. These results argue that the tif-1 mutation (or mutations) alters the DNA binding site of the recA protein. We suggest that in vivo the tif(recA) protein is activated for cleaving repressors of SOS genes by complex formation with short single-stranded regions or gaps that normally occur near the growing fork of replicating chromosomes and are too short for activating the recA+ enzyme. This mechanism can account for the expression of SOS functions in the absence of DNA damage in tif mutant strains. Images PMID:7031642

Role of co-regulators in metabolic and transcriptional actions of thyroid hormone.

PubMed

Astapova, Inna

2016-04-01

Thyroid hormone (TH) controls a wide range of physiological processes through TH receptor (TR) isoforms. Classically, TRs are proposed to function as tri-iodothyronine (T3)-dependent transcription factors: on positively regulated target genes, unliganded TRs mediate transcriptional repression through recruitment of co-repressor complexes, while T3 binding leads to dismissal of co-repressors and recruitment of co-activators to activate transcription. Co-repressors and co-activators were proposed to play opposite roles in the regulation of negative T3 target genes and hypothalamic-pituitary-thyroid axis, but exact mechanisms of the negative regulation by TH have remained elusive. Important insights into the roles of co-repressors and co-activators in different physiological processes have been obtained using animal models with disrupted co-regulator function. At the same time, recent studies interrogating genome-wide TR binding have generated compelling new data regarding effects of T3, local chromatin structure, and specific response element configuration on TR recruitment and function leading to the proposal of new models of transcriptional regulation by TRs. This review discusses data obtained in various mouse models with manipulated function of nuclear receptor co-repressor (NCoR or NCOR1) and silencing mediator of retinoic acid receptor and thyroid hormone receptor (SMRT or NCOR2), and family of steroid receptor co-activators (SRCs also known as NCOAs) in the context of TH action, as well as insights into the function of co-regulators that may emerge from the genome-wide TR recruitment analysis. © 2016 Society for Endocrinology.

Integrative genome-wide analysis reveals HLP1, a novel RNA-binding protein, regulates plant flowering by targeting alternative polyadenylation

PubMed Central

Zhang, Yong; Gu, Lianfeng; Hou, Yifeng; Wang, Lulu; Deng, Xian; Hang, Runlai; Chen, Dong; Zhang, Xiansheng; Zhang, Yi; Liu, Chunyan; Cao, Xiaofeng

2015-01-01

Alternative polyadenylation (APA) is a widespread mechanism for gene regulation and has been implicated in flowering, but the molecular basis governing the choice of a specific poly(A) site during the vegetative-to-reproductive growth transition remains unclear. Here we characterize HLP1, an hnRNP A/B protein as a novel regulator for pre-mRNA 3′-end processing in Arabidopsis. Genetic analysis reveals that HLP1 suppresses Flowering Locus C (FLC), a key repressor of flowering in Arabidopsis. Genome-wide mapping of HLP1-RNA interactions indicates that HLP1 binds preferentially to A-rich and U-rich elements around cleavage and polyadenylation sites, implicating its role in 3′-end formation. We show HLP1 is significantly enriched at transcripts involved in RNA metabolism and flowering. Comprehensive profiling of the poly(A) site usage reveals that HLP1 mutations cause thousands of poly(A) site shifts. A distal-to-proximal poly(A) site shift in the flowering regulator FCA, a direct target of HLP1, leads to upregulation of FLC and delayed flowering. Our results elucidate that HLP1 is a novel factor involved in 3′-end processing and controls reproductive timing via targeting APA. PMID:26099751

A regulatory network to segregate the identity of neuronal subtypes.

PubMed

Lee, Seunghee; Lee, Bora; Joshi, Kaumudi; Pfaff, Samuel L; Lee, Jae W; Lee, Soo-Kyung

2008-06-01

Spinal motor neurons (MNs) and V2 interneurons (V2-INs) are specified by two related LIM-complexes, MN-hexamer and V2-tetramer, respectively. Here we show how multiple parallel and complementary feedback loops are integrated to assign these two cell fates accurately. While MN-hexamer response elements (REs) are specific to MN-hexamer, V2-tetramer-REs can bind both LIM-complexes. In embryonic MNs, however, two factors cooperatively suppress the aberrant activation of V2-tetramer-REs. First, LMO4 blocks V2-tetramer assembly. Second, MN-hexamer induces a repressor, Hb9, which binds V2-tetramer-REs and suppresses their activation. V2-INs use a similar approach; V2-tetramer induces a repressor, Chx10, which binds MN-hexamer-REs and blocks their activation. Thus, our study uncovers a regulatory network to segregate related cell fates, which involves reciprocal feedforward gene regulatory loops.

Investigation of arc repressor DNA-binding specificity by comparative molecular dynamics simulations.

PubMed

Song, Wei; Guo, Jun-Tao

2015-01-01

Transcription factors regulate gene expression through binding to specific DNA sequences. How transcription factors achieve high binding specificity is still not well understood. In this paper, we investigated the role of protein flexibility in protein-DNA-binding specificity by comparative molecular dynamics (MD) simulations. Protein flexibility has been considered as a key factor in molecular recognition, which is intrinsically a dynamic process involving fine structural fitting between binding components. In this study, we performed comparative MD simulations on wild-type and F10V mutant P22 Arc repressor in both free and complex conformations. The F10V mutant has lower DNA-binding specificity though both the bound and unbound main-chain structures between the wild-type and F10V mutant Arc are highly similar. We found that the DNA-binding motif of wild-type Arc is structurally more flexible than the F10V mutant in the unbound state, especially for the six DNA base-contacting residues in each dimer. We demonstrated that the flexible side chains of wild-type Arc lead to a higher DNA-binding specificity through forming more hydrogen bonds with DNA bases upon binding. Our simulations also showed a possible conformational selection mechanism for Arc-DNA binding. These results indicate the important roles of protein flexibility and dynamic properties in protein-DNA-binding specificity.

CpG methylation of HPV 16 LCR at E2 binding site proximal to P97 is associated with cervical cancer in presence of intact E2.

PubMed

Bhattacharjee, Bornali; Sengupta, Sharmila

2006-10-25

Human papillomavirus type 16 (HPV-16) E2 protein negatively regulates transcription of the E6 and E7 genes. This study was done to test the hypothesis that methylation of the HPV 16 long control region (LCR) is overrepresented among cervical cancer (CaCx) cases compared to cytologically normal controls harboring intact E2 gene. Methylation of the E2 binding site (E2BS-I), proximal to the P97 promoter, was assessed by HpaII/ MspI restriction digestion while McrBC digestion was used to assess LCR-E6 (7289-540) for 57 CaCx samples and 15 normal controls. E2BS-I methylation was found to be significantly higher (56.14%) in cases compared to (20%) controls [OR(age-adjusted) (95% CI): 4.53 (1.05-19.43) p=0.042]. The difference between cases (54.39%) and controls (40%) with respect to LCR-E6 methylation status [OR(age-adjusted) (95% CI): 1.77(0.5-6.3); p=0.38] was not significant. Sequencing of a randomly selected set of 13 methylated malignant samples revealed absence or rare presence, of methylation at CpGs 7579, 7535, 7683 and 7862 respectively. Methylation was found to be more at CpGs within E2 binding sites proximal to the P97 promoter. These results indicate the involvement of E2 binding site methylation in presence of intact E2, leading to loss of E2 repressor activity in CaCx.

LexA Binds to Transcription Regulatory Site of Cell Division Gene ftsZ in Toxic Cyanobacterium Microcystis aeruginosa.

PubMed

Honda, Takashi; Morimoto, Daichi; Sako, Yoshihiko; Yoshida, Takashi

2018-05-17

Previously, we showed that DNA replication and cell division in toxic cyanobacterium Microcystis aeruginosa are coordinated by transcriptional regulation of cell division gene ftsZ and that an unknown protein specifically bound upstream of ftsZ (BpFz; DNA-binding protein to an upstream site of ftsZ) during successful DNA replication and cell division. Here, we purified BpFz from M. aeruginosa strain NIES-298 using DNA-affinity chromatography and gel-slicing combined with gel electrophoresis mobility shift assay (EMSA). The N-terminal amino acid sequence of BpFz was identified as TNLESLTQ, which was identical to that of transcription repressor LexA from NIES-843. EMSA analysis using mutant probes showed that the sequence GTACTAN 3 GTGTTC was important in LexA binding. Comparison of the upstream regions of lexA in the genomes of closely related cyanobacteria suggested that the sequence TASTRNNNNTGTWC could be a putative LexA recognition sequence (LexA box). Searches for TASTRNNNNTGTWC as a transcriptional regulatory site (TRS) in the genome of M. aeruginosa NIES-843 showed that it was present in genes involved in cell division, photosynthesis, and extracellular polysaccharide biosynthesis. Considering that BpFz binds to the TRS of ftsZ during normal cell division, LexA may function as a transcriptional activator of genes related to cell reproduction in M. aeruginosa, including ftsZ. This may be an example of informality in the control of bacterial cell division.

Cell cycle-dependent transcription factors control the expression of yeast telomerase RNA.

PubMed

Dionne, Isabelle; Larose, Stéphanie; Dandjinou, Alain T; Abou Elela, Sherif; Wellinger, Raymund J

2013-07-01

Telomerase is a specialized ribonucleoprotein that adds repeated DNA sequences to the ends of eukaryotic chromosomes to preserve genome integrity. Some secondary structure features of the telomerase RNA are very well conserved, and it serves as a central scaffold for the binding of associated proteins. The Saccharomyces cerevisiae telomerase RNA, TLC1, is found in very low copy number in the cell and is the limiting component of the known telomerase holoenzyme constituents. The reasons for this low abundance are unclear, but given that the RNA is very stable, transcriptional control mechanisms must be extremely important. Here we define the sequences forming the TLC1 promoter and identify the elements required for its low expression level, including enhancer and repressor elements. Within an enhancer element, we found consensus sites for Mbp1/Swi4 association, and chromatin immunoprecipitation (ChIP) assays confirmed the binding of Mbp1 and Swi4 to these sites of the TLC1 promoter. Furthermore, the enhancer element conferred cell cycle-dependent regulation to a reporter gene, and mutations in the Mbp1/Swi4 binding sites affected the levels of telomerase RNA and telomere length. Finally, ChIP experiments using a TLC1 RNA-binding protein as target showed cell cycle-dependent transcription of the TLC1 gene. These results indicate that the budding yeast TLC1 RNA is transcribed in a cell cycle-dependent fashion late in G1 and may be part of the S phase-regulated group of genes involved in DNA replication.

Assessing the Role of ETHYLENE RESPONSE FACTOR Transcriptional Repressors in Salicylic Acid-Mediated Suppression of Jasmonic Acid-Responsive Genes.

PubMed

Caarls, Lotte; Van der Does, Dieuwertje; Hickman, Richard; Jansen, Wouter; Verk, Marcel C Van; Proietti, Silvia; Lorenzo, Oscar; Solano, Roberto; Pieterse, Corné M J; Van Wees, Saskia C M

2017-02-01

Salicylic acid (SA) and jasmonic acid (JA) cross-communicate in the plant immune signaling network to finely regulate induced defenses. In Arabidopsis, SA antagonizes many JA-responsive genes, partly by targeting the ETHYLENE RESPONSE FACTOR (ERF)-type transcriptional activator ORA59. Members of the ERF transcription factor family typically bind to GCC-box motifs in the promoters of JA- and ethylene-responsive genes, thereby positively or negatively regulating their expression. The GCC-box motif is sufficient for SA-mediated suppression of JA-responsive gene expression. Here, we investigated whether SA-induced ERF-type transcriptional repressors, which may compete with JA-induced ERF-type activators for binding at the GCC-box, play a role in SA/JA antagonism. We selected ERFs that are transcriptionally induced by SA and/or possess an EAR transcriptional repressor motif. Several of the 16 ERFs tested suppressed JA-dependent gene expression, as revealed by enhanced JA-induced PDF1.2 or VSP2 expression levels in the corresponding erf mutants, while others were involved in activation of these genes. However, SA could antagonize JA-induced PDF1.2 or VSP2 in all erf mutants, suggesting that the tested ERF transcriptional repressors are not required for SA/JA cross-talk. Moreover, a mutant in the co-repressor TOPLESS, that showed reduction in repression of JA signaling, still displayed SA-mediated antagonism of PDF1.2 and VSP2. Collectively, these results suggest that SA-regulated ERF transcriptional repressors are not essential for antagonism of JA-responsive gene expression by SA. We further show that de novo SA-induced protein synthesis is required for suppression of JA-induced PDF1.2, pointing to SA-stimulated production of an as yet unknown protein that suppresses JA-induced transcription. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The Verrucomicrobia LexA-Binding Motif: Insights into the Evolutionary Dynamics of the SOS Response.

PubMed

Erill, Ivan; Campoy, Susana; Kılıç, Sefa; Barbé, Jordi

2016-01-01

The SOS response is the primary bacterial mechanism to address DNA damage, coordinating multiple cellular processes that include DNA repair, cell division, and translesion synthesis. In contrast to other regulatory systems, the composition of the SOS genetic network and the binding motif of its transcriptional repressor, LexA, have been shown to vary greatly across bacterial clades, making it an ideal system to study the co-evolution of transcription factors and their regulons. Leveraging comparative genomics approaches and prior knowledge on the core SOS regulon, here we define the binding motif of the Verrucomicrobia, a recently described phylum of emerging interest due to its association with eukaryotic hosts. Site directed mutagenesis of the Verrucomicrobium spinosum recA promoter confirms that LexA binds a 14 bp palindromic motif with consensus sequence TGTTC-N4-GAACA. Computational analyses suggest that recognition of this novel motif is determined primarily by changes in base-contacting residues of the third alpha helix of the LexA helix-turn-helix DNA binding motif. In conjunction with comparative genomics analysis of the LexA regulon in the Verrucomicrobia phylum, electrophoretic shift assays reveal that LexA binds to operators in the promoter region of DNA repair genes and a mutagenesis cassette in this organism, and identify previously unreported components of the SOS response. The identification of tandem LexA-binding sites generating instances of other LexA-binding motifs in the lexA gene promoter of Verrucomicrobia species leads us to postulate a novel mechanism for LexA-binding motif evolution. This model, based on gene duplication, successfully addresses outstanding questions in the intricate co-evolution of the LexA protein, its binding motif and the regulatory network it controls.

Involvement of Sp1 and Microsatellite Repressor Sequences in the Transcriptional Control of the Human CD30 Gene

PubMed Central

Croager, Emma J.; Gout, Alexander M.; Abraham, Lawrence J.

2000-01-01

CD30, as a member of the tumor necrosis factor (TNF) receptor family, is expressed on the surface of activated lymphoid cells. CD30 overexpression is a characteristic of lymphoproliferative diseases such as Hodgkin’s/non-Hodgkin’s lymphomas, embryonal carcinoma, and a number of Th2-associated diseases. The CD30 gene has been mapped to a region of the murine genome that is involved in susceptibility to systemic lupus erythematosus. Functionally, CD30 may play a role in the deletion of autoreactive T cells. We were interested in determining the molecular nature of CD30 overexpression. Sequence comparison has revealed significant identity between the TATA-less human and murine CD30 promoters; they share a number of common consensus binding motifs. Transfection assays identified three regions of transcriptional importance; the region between position −1.2 kb and −336 bp, containing a CCAT microsatellite sequence, a conserved Sp1 site at positions −43 to −38, and a downstream promoter element (DPE) at positions +24 to +29. EMSA and DNase I footprinting showed specific DNA-protein interactions of the CD30 promoter with the Sp1 site and the CCAT repeat region. The DPE element was shown to be essential for start site selection. We conclude that the conserved Sp1 site at −43 to −38 is associated with maximum reporter gene activity, the DPE element is required for start site selection, and the CCAT tetranucleotide repeats act to repress transcription. We also have shown that the microsatellite is multiallelic, when we screened a random healthy population. Further studies are required to determine whether microsatellite instability in the repressor predisposes susceptible individuals to CD30 overexpression. PMID:10793083

Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer

PubMed Central

Struffi, Paolo; Corado, Maria; Kaplan, Leah; Yu, Danyang; Rushlow, Christine; Small, Stephen

2011-01-01

Despite years of study, the precise mechanisms that control position-specific gene expression during development are not understood. Here, we analyze an enhancer element from the even skipped (eve) gene, which activates and positions two stripes of expression (stripes 3 and 7) in blastoderm stage Drosophila embryos. Previous genetic studies showed that the JAK-STAT pathway is required for full activation of the enhancer, whereas the gap genes hunchback (hb) and knirps (kni) are required for placement of the boundaries of both stripes. We show that the maternal zinc-finger protein Zelda (Zld) is absolutely required for activation, and present evidence that Zld binds to multiple non-canonical sites. We also use a combination of in vitro binding experiments and bioinformatics analysis to redefine the Kni-binding motif, and mutational analysis and in vivo tests to show that Kni and Hb are dedicated repressors that function by direct DNA binding. These experiments significantly extend our understanding of how the eve enhancer integrates positive and negative transcriptional activities to generate sharp boundaries in the early embryo. PMID:21865322

E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism.

PubMed Central

Robert-Le Meur, M; Portier, C

1992-01-01

It has been previously shown that the pnp messenger RNAs are cleaved by RNase III at the 5' end and that these cleavages induce a rapid decay of these messengers. A translational fusion between pnp and lacZ was introduced into the chromosome of a delta lac strain to study the expression of pnp. In the presence of increased cellular concentrations of polynucleotide phosphorylase, the level of the hybrid beta-galactosidase is repressed, whereas the synthesis rate of the corresponding message is not significantly affected. In the absence of pnp, the level of the hybrid protein increases strongly. Thus, polynucleotide phosphorylase is post-transcriptionally autocontrolled. However, autocontrol is totally abolished in strains where the RNase III site on the pnp message has been deleted or in strains devoid of RNase III. These results suggest that polynucleotide phosphorylase requires RNase III cleavages to autoregulate the translation of its message. Other mutations in the ribosome binding site region support the hypothesis that this 3' to 5' processive enzyme could recognize a specific repressor binding site at the 5' end of pnp mRNA. Implications of these results on the mechanism of regulation and on messenger degradation are discussed. Images PMID:1628624

E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism.

PubMed

Robert-Le Meur, M; Portier, C

1992-07-01

It has been previously shown that the pnp messenger RNAs are cleaved by RNase III at the 5' end and that these cleavages induce a rapid decay of these messengers. A translational fusion between pnp and lacZ was introduced into the chromosome of a delta lac strain to study the expression of pnp. In the presence of increased cellular concentrations of polynucleotide phosphorylase, the level of the hybrid beta-galactosidase is repressed, whereas the synthesis rate of the corresponding message is not significantly affected. In the absence of pnp, the level of the hybrid protein increases strongly. Thus, polynucleotide phosphorylase is post-transcriptionally autocontrolled. However, autocontrol is totally abolished in strains where the RNase III site on the pnp message has been deleted or in strains devoid of RNase III. These results suggest that polynucleotide phosphorylase requires RNase III cleavages to autoregulate the translation of its message. Other mutations in the ribosome binding site region support the hypothesis that this 3' to 5' processive enzyme could recognize a specific repressor binding site at the 5' end of pnp mRNA. Implications of these results on the mechanism of regulation and on messenger degradation are discussed.

Crystallization and preliminary X-ray diffraction analysis of the arginine repressor of the hyperthermophile Thermotoga neapolitana

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

Massant, Jan, E-mail: jan.massant@vub.ac.be; Peeters, Eveline; Charlier, Daniel

2006-01-01

The arginine repressor of the hyperthermophile T. neapolitana was crystallized with and without its corepressor arginine. Both crystals diffracted to high resolution and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with similar unit-cell parameters. The arginine repressor of Thermotoga neapolitana (ArgRTnp) is a member of the family of multifunctional bacterial arginine repressors involved in the regulation of arginine metabolism. This hyperthermophilic repressor shows unique DNA-binding features that distinguish it from its homologues. ArgRTnp exists as a homotrimeric protein that assembles into hexamers at higher protein concentrations and/or in the presence of arginine. ArgRTnp was crystallized with andmore » without its corepressor arginine using the hanging-drop vapour-diffusion method. Crystals of the aporepressor diffracted to a resolution of 2.1 Å and belong to the orthorhombic P2{sub 1}2{sub 1}2{sub 1} space group, with unit-cell parameters a = 117.73, b = 134.15, c = 139.31 Å. Crystals of the repressor in the presence of its corepressor arginine diffracted to a resolution of 2.4 Å and belong to the same space group, with similar unit-cell parameters.« less

Structural insights into selective agonist actions of tamoxifen on human estrogen receptor alpha.

PubMed

Chakraborty, Sandipan; Biswas, Pradip Kumar

2014-08-01

Tamoxifen-an anti-estrogenic ligand in breast tissues used as a first-line treatment in estrogen receptor (ER)-positive breast cancers-is associated with the development of resistance followed by resumption of tumor growth in about 30 % of cases. Whether tamoxifen assists in proliferation in such cases or whether any ligand-independent pathway to transcription exists is not fully understood; also, no ERα mutants have been detected so far that could lead to tamoxifen resistance. Using in silico conformational analysis of the ERα ligand binding domain (LBD), in the absence and presence of selective agonist (diethylstilbestrol; DES), antagonist (Faslodex; ICI), and selective estrogen receptor modulator (SERM; 4-hydroxy tamoxifen; 4-OHT) ligands, we have elucidated ligand-responsive structural modulations of the ERα-LBD dimer in its agonist and antagonist complexes to address the issue of "tamoxifen resistance". DES and ICI were found to stabilize the dimer in their agonist and antagonist conformations, respectively. The ERα-LBD dimer without the presence of any bound ligand also led to a stable structure in agonist conformation. However, binding of 4-OHT to the antagonist structure led to a flexible conformation allowing the protein to visit conformations populated by agonists as was evident from principal component analysis and radius of gyration plots. Further, the relaxed conformations of the 4-OHT bound protein exhibited a diminished size of the co-repressor binding pocket in the LBD, thus signaling a partial blockage of the co-repressor binding motif. Thus, the ability of 4-OHT-bound ERα-LBD to assume flexible conformations visited by agonists and reduced co-repressor binding surface at the LBD provide crucial structural insights into tamoxifen-resistance that complement our existing understanding.

Effects of cytosine methylation on transcription factor binding sites

PubMed Central

2014-01-01

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

Electrostatic occlusion and quaternary structural ion pairing are key determinants of Cu(I)-mediated allostery in the copper-sensing operon repressor (CsoR).

PubMed

Chang, Feng-Ming James; Martin, Julia E; Giedroc, David P

2015-04-21

The copper-sensing operon repressor (CsoR) is an all-α-helical disc-shaped D2-symmetric homotetramer that forms a 2:1 tetramer/DNA operator complex and represses the expression of copper-resistance genes in a number of bacteria. A previous bioinformatics analysis of CsoR-family repressors distributes Cu(I)-sensing CsoRs in four of seven distinct clades on the basis of global sequence similarity. In this work, we define energetically important determinants of DNA binding in the apo-state (ΔΔGbind), and for allosteric negative coupling of Cu(I) binding to DNA binding (ΔΔGc) in a model clade IV CsoR from Geobacillus thermodenitrificans (Gt) of known structure, by selectively targeting for mutagenesis those charged residues uniquely conserved in clade IV CsoRs. These include a folded N-terminal "tail" and a number of Cu(I)-sensor and clade-specific residues that when mapped onto a model of Cu(I)-bound Gt CsoR define a path across one face of the tetramer. We find that Cu(I)-binding prevents formation of the 2:1 "sandwich" complex rather than DNA binding altogether. Folding of the N-terminal tail (residues R18, E22, R74) upon Cu-binding to the periphery of the tetramer inhibits assembly of the 2:1 apoprotein-DNA complex. In contrast, Ala substitution of residues that surround the central "hole" (R65, K101) in the tetramer, as well R48, impact DNA binding. We also identify a quaternary structural ion-pair, E73-K101″, that crosses the tetramer interface, charge-reversal of which restores DNA binding activity, allosteric regulation by Cu(I), and transcriptional derepression by Cu(I) in cells. These findings suggest an "electrostatic occlusion" model, in which basic residues important for DNA binding and/or allostery become sequestered via ion-pairing specifically in the Cu(I)-bound state, and this aids in copper-dependent disassembly of a repression complex.

Dissecting the link between the enzymatic activity and the SaPI inducing capacity of the phage 80α dUTPase.

PubMed

Alite, Christian; Humphrey, Suzanne; Donderis, Jordi; Maiques, Elisa; Ciges-Tomas, J Rafael; Penadés, José R; Marina, Alberto

2017-09-11

The trimeric staphylococcal phage-encoded dUTPases (Duts) are signalling molecules that induce the cycle of some Staphylococcal pathogenicity islands (SaPIs) by binding to the SaPI-encoded Stl repressor. To perform this regulatory role, these Duts require an extra motif VI, as well as the Dut conserved motifs IV and V. While the apo form of Dut is required for the interaction with the Stl repressor, usually only those Duts with normal enzymatic activity can induce the SaPI cycle. To understand the link between the enzymatic activities and inducing capacities of the Dut protein, we analysed the structural, biochemical and physiological characteristics of the Dut80α D95E mutant, which loses the SaPI cycle induction capacity despite retaining enzymatic activity. Asp95 is located at the threefold central channel of the trimeric Dut where it chelates a divalent ion. Here, using state-of-the-art techniques, we demonstrate that D95E mutation has an epistatic effect on the motifs involved in Stl binding. Thus, ion binding in the central channel correlates with the capacity of motif V to twist and order in the SaPI-inducing disposition, while the tip of motif VI is disturbed. These alterations in turn reduce the affinity for the Stl repressor and the capacity to induce the SaPI cycle.

A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction

PubMed Central

Tsai, Kuang-Lei; Sato, Shigeo; Tomomori-Sato, Chieri; Conaway, Ronald C.; Conaway, Joan W.; Asturias, Francisco J.

2013-01-01

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a preeminent complex in eukaryotic transcription regulation. We used macromolecular electron microscopy and biochemistry to investigate the subunit organization, structure, and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator’s Middle module interferes with CTD-dependent RNAPII binding to a previously unknown Middle module CTD-binding site targeted early on in a multi-step holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD, and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the Mediator mechanism. PMID:23563140

FoxG1 and TLE2 act cooperatively to regulate ventral telencephalon formation.

PubMed

Roth, Martin; Bonev, Boyan; Lindsay, Jennefer; Lea, Robert; Panagiotaki, Niki; Houart, Corinne; Papalopulu, Nancy

2010-05-01

FoxG1 is a conserved transcriptional repressor that plays a key role in the specification, proliferation and differentiation of the telencephalon, and is expressed from the earliest stages of telencephalic development through to the adult. How the interaction with co-factors might influence the multiplicity and diversity of FoxG1 function is not known. Here, we show that interaction of FoxG1 with TLE2, a Xenopus tropicalis co-repressor of the Groucho/TLE family, is crucial for regulating the early activity of FoxG1. We show that TLE2 is co-expressed with FoxG1 in the ventral telencephalon from the early neural plate stage and functionally cooperates with FoxG1 in an ectopic neurogenesis assay. FoxG1 has two potential TLE binding sites: an N-terminal eh1 motif and a C-terminal YWPMSPF motif. Although direct binding seems to be mediated by the N-terminal motif, both motifs appear important for functional synergism. In the neurogenesis assay, mutation of either motif abolishes functional cooperation of TLE2 with FoxG1, whereas in the forebrain deletion of both motifs renders FoxG1 unable to induce the ventral telencephalic marker Nkx2.1. Knocking down either FoxG1 or TLE2 disrupts the development of the ventral telencephalon, supporting the idea that endogenous TLE2 and FoxG1 work together to specify the ventral telencephalon.

FoxG1 and TLE2 act cooperatively to regulate ventral telencephalon formation

PubMed Central

Roth, Martin; Bonev, Boyan; Lindsay, Jennefer; Lea, Robert; Panagiotaki, Niki; Houart, Corinne; Papalopulu, Nancy

2010-01-01

FoxG1 is a conserved transcriptional repressor that plays a key role in the specification, proliferation and differentiation of the telencephalon, and is expressed from the earliest stages of telencephalic development through to the adult. How the interaction with co-factors might influence the multiplicity and diversity of FoxG1 function is not known. Here, we show that interaction of FoxG1 with TLE2, a Xenopus tropicalis co-repressor of the Groucho/TLE family, is crucial for regulating the early activity of FoxG1. We show that TLE2 is co-expressed with FoxG1 in the ventral telencephalon from the early neural plate stage and functionally cooperates with FoxG1 in an ectopic neurogenesis assay. FoxG1 has two potential TLE binding sites: an N-terminal eh1 motif and a C-terminal YWPMSPF motif. Although direct binding seems to be mediated by the N-terminal motif, both motifs appear important for functional synergism. In the neurogenesis assay, mutation of either motif abolishes functional cooperation of TLE2 with FoxG1, whereas in the forebrain deletion of both motifs renders FoxG1 unable to induce the ventral telencephalic marker Nkx2.1. Knocking down either FoxG1 or TLE2 disrupts the development of the ventral telencephalon, supporting the idea that endogenous TLE2 and FoxG1 work together to specify the ventral telencephalon. PMID:20356955

Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.

PubMed

Grossoehme, Nicholas; Kehl-Fie, Thomas E; Ma, Zhen; Adams, Keith W; Cowart, Darin M; Scott, Robert A; Skaar, Eric P; Giedroc, David P

2011-04-15

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027-0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes.

Control of Copper Resistance and Inorganic Sulfur Metabolism by Paralogous Regulators in Staphylococcus aureus*

PubMed Central

Grossoehme, Nicholas; Kehl-Fie, Thomas E.; Ma, Zhen; Adams, Keith W.; Cowart, Darin M.; Scott, Robert A.; Skaar, Eric P.; Giedroc, David P.

2011-01-01

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027–0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes. PMID:21339296

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

Oh, Yohan; Chung, Kwang Chul, E-mail: kchung@yonsei.ac.kr

Highlights: Black-Right-Pointing-Pointer ZNF131 directly interacts with ER{alpha}. Black-Right-Pointing-Pointer The binding affinity of ZNF131 to ER{alpha} increases upon E2 stimulation. Black-Right-Pointing-Pointer ZNF131 inhibits ER{alpha}-mediated trans-activation by suppressing its homo-dimerization. Black-Right-Pointing-Pointer ZNF131 inhibits ER{alpha}-dimerization and E2-induced breast cancer cell proliferation. Black-Right-Pointing-Pointer ZNF131 inhibits estrogen signaling by acting as an ER{alpha}-co-repressor. -- Abstract: Steroid hormone estrogen elicits various physiological functions, many of which are mediated through two structurally and functionally distinct estrogen receptors, ER{alpha} and ER{beta}. The functional role of zinc finger protein 131 (ZNF131) is poorly understood, but it is assumed to possess transcriptional regulation activity due to the presence of amore » DNA binding motif. A few recent reports, including ours, revealed that ZNF131 acts as a negative regulator of ER{alpha} and that SUMO modification potentiates the negative effect of ZNF131 on estrogen signaling. However, its molecular mechanism for ER{alpha} inhibition has not been elucidated in detail. Here, we demonstrate that ZNF131 directly interacts with ER{alpha}, which consequently inhibits ER{alpha}-mediated trans-activation by suppressing its homo-dimerization. Moreover, we show that the C-terminal region of ZNF131 containing the SUMOylation site is necessary for its inhibition of estrogen signaling. Taken together, these data suggest that ZNF131 inhibits estrogen signaling by acting as an ER{alpha}-co-repressor.« less

Specification of skeletal muscle differentiation by repressor element-1 silencing transcription factor (REST)-regulated Kv7.4 potassium channels

PubMed Central

Iannotti, Fabio Arturo; Barrese, Vincenzo; Formisano, Luigi; Miceli, Francesco; Taglialatela, Maurizio

2013-01-01

Changes in the expression of potassium (K+) channels is a pivotal event during skeletal muscle differentiation. In mouse C2C12 cells, similarly to human skeletal muscle cells, myotube formation increased the expression of Kv7.1, Kv7.3, and Kv7.4, the last showing the highest degree of regulation. In C2C12 cells, Kv7.4 silencing by RNA interference reduced the expression levels of differentiation markers (myogenin, myosin heavy chain, troponinT-1, and Pax3) and impaired myotube formation and multinucleation. In Kv7.4-silenced cells, the differentiation-promoting effect of the Kv7 activator N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester (retigabine) was abrogated. Expression levels for the repressor element-1 silencing transcription factor (REST) declined during myotube formation. Transcript levels for Kv7.4, as well as for myogenin, troponinT-1, and Pax3, were reduced by REST overexpression and enhanced upon REST suppression by RNA interference. Four regions containing potential REST-binding sites in the 5′ untranslated region and in the first intron of the Kv7.4 gene were identified by bioinformatic analysis. Chromatin immunoprecipitation assays showed that REST binds to these regions, exhibiting a higher efficiency in myoblasts than in myotubes. These data suggest that Kv7.4 plays a permissive role in skeletal muscle differentiation and highlight REST as a crucial transcriptional regulator for this K+ channel subunit. PMID:23242999

Efficient analysis of stochastic gene dynamics in the non-adiabatic regime using piecewise deterministic Markov processes

PubMed Central

2018-01-01

Single-cell experiments show that gene expression is stochastic and bursty, a feature that can emerge from slow switching between promoter states with different activities. In addition to slow chromatin and/or DNA looping dynamics, one source of long-lived promoter states is the slow binding and unbinding kinetics of transcription factors to promoters, i.e. the non-adiabatic binding regime. Here, we introduce a simple analytical framework, known as a piecewise deterministic Markov process (PDMP), that accurately describes the stochastic dynamics of gene expression in the non-adiabatic regime. We illustrate the utility of the PDMP on a non-trivial dynamical system by analysing the properties of a titration-based oscillator in the non-adiabatic limit. We first show how to transform the underlying chemical master equation into a PDMP where the slow transitions between promoter states are stochastic, but whose rates depend upon the faster deterministic dynamics of the transcription factors regulated by these promoters. We show that the PDMP accurately describes the observed periods of stochastic cycles in activator and repressor-based titration oscillators. We then generalize our PDMP analysis to more complicated versions of titration-based oscillators to explain how multiple binding sites lengthen the period and improve coherence. Last, we show how noise-induced oscillation previously observed in a titration-based oscillator arises from non-adiabatic and discrete binding events at the promoter site. PMID:29386401

Heterodimerization of Msx and Dlx homeoproteins results in functional antagonism.

PubMed

Zhang, H; Hu, G; Wang, H; Sciavolino, P; Iler, N; Shen, M M; Abate-Shen, C

1997-05-01

Protein-protein interactions are known to be essential for specifying the transcriptional activities of homeoproteins. Here we show that representative members of the Msx and Dlx homeoprotein families form homo- and heterodimeric complexes. We demonstrate that dimerization by Msx and Dlx proteins is mediated through their homeodomains and that the residues required for this interaction correspond to those necessary for DNA binding. Unlike most other known examples of homeoprotein interactions, association of Msx and Dlx proteins does not promote cooperative DNA binding; instead, dimerization and DNA binding are mutually exclusive activities. In particular, we show that Msx and Dlx proteins interact independently and noncooperatively with homeodomain DNA binding sites and that dimerization is specifically blocked by the presence of such DNA sites. We further demonstrate that the transcriptional properties of Msx and Dlx proteins display reciprocal inhibition. Specifically, Msx proteins act as transcriptional repressors and Dlx proteins act as activators, while in combination, Msx and Dlx proteins counteract each other's transcriptional activities. Finally, we show that the expression patterns of representative Msx and Dlx genes (Msx1, Msx2, Dlx2, and Dlx5) overlap in mouse embryogenesis during limb bud and craniofacial development, consistent with the potential for their protein products to interact in vivo. Based on these observations, we propose that functional antagonism through heterodimer formation provides a mechanism for regulating the transcriptional actions of Msx and Dlx homeoproteins in vivo.

Expression, purification and preliminary X-ray analysis of the C-terminal domain of an arginine repressor protein from Mycobacterium tuberculosis

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

Lu, George J.; Garen, Craig R.; Cherney, Maia M.

2007-11-01

The C-terminal portion of the arginine repressor protein from M. tuberculosis H37Rv has been crystallized. The complete transcriptional factor regulates arginine biosynthesis by binding operator DNA when arginine is bound at the C-terminal domain. 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 Å. The crystals belong to space group P1 and themore » 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.« less

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

The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110.

PubMed

Wolf, Timo; Droste, Julian; Gren, Tetiana; Ortseifen, Vera; Schneiker-Bekel, Susanne; Zemke, Till; Pühler, Alfred; Kalinowski, Jörn

2017-07-25

Acarbose is used in the treatment of diabetes mellitus type II and is produced by Actinoplanes sp. SE50/110. Although the biosynthesis of acarbose has been intensively studied, profound knowledge about transcription factors involved in acarbose biosynthesis and their binding sites has been missing until now. In contrast to acarbose biosynthetic gene clusters in Streptomyces spp., the corresponding gene cluster of Actinoplanes sp. SE50/110 lacks genes for transcriptional regulators. The acarbose regulator C (AcrC) was identified through an in silico approach by aligning the LacI family regulators of acarbose biosynthetic gene clusters in Streptomyces spp. with the Actinoplanes sp. SE50/110 genome. The gene for acrC, located in a head-to-head arrangement with the maltose/maltodextrin ABC transporter malEFG operon, was deleted by introducing PCR targeting for Actinoplanes sp. SE50/110. Characterization was carried out through cultivation experiments, genome-wide microarray hybridizations, and RT-qPCR as well as electrophoretic mobility shift assays for the elucidation of binding motifs. The results show that AcrC binds to the intergenic region between acbE and acbD in Actinoplanes sp. SE50/110 and acts as a transcriptional repressor on these genes. The transcriptomic profile of the wild type was reconstituted through a complementation of the deleted acrC gene. Additionally, regulatory sequence motifs for the binding of AcrC were identified in the intergenic region of acbE and acbD. It was shown that AcrC expression influences acarbose formation in the early growth phase. Interestingly, AcrC does not regulate the malEFG operon. This study characterizes the first known transcription factor of the acarbose biosynthetic gene cluster in Actinoplanes sp. SE50/110. It therefore represents an important step for understanding the regulatory network of this organism. Based on this work, rational strain design for improving the biotechnological production of acarbose can now be implemented.

An exclusive α/β code directs allostery in TetR-peptide complexes.

PubMed

Sevvana, Madhumati; Goetz, Christoph; Goeke, Dagmar; Wimmer, Cornelius; Berens, Christian; Hillen, Wolfgang; Muller, Yves A

2012-02-10

The allosteric mechanism of one of the best characterized bacterial transcription regulators, tetracycline repressor (TetR), has recently been questioned. Tetracycline binding induces cooperative folding of TetR, as suggested by recent unfolding studies, rather than switching between two defined conformational states, namely a DNA-binding-competent conformation and a non-DNA-binding conformation. Upon ligand binding, a host of near-native multiconformational structures collapse into a single, highly stabilized protein conformation that is no longer able to bind DNA. Here, structure-function studies performed with four synthetic peptides that bind to TetR and mimic the function of low-molecular-weight effectors, such as tetracyclines, provide new means to discriminate between different allosteric models. Whereas two inducing peptides bind in an extended β-like conformation, two anti-inducing peptides form an α-helix in the effector binding site of TetR. This exclusive bimodal interaction mode coincides with two distinct overall conformations of TetR, namely one that is identical with induced TetR and one that mirrors the DNA-bound state of TetR. Urea-induced unfolding studies show no increase in thermodynamic stability for any of the peptide complexes, although fluorescence measurements demonstrate peptide binding to TetR. This strongly suggests that, at least for these peptide effectors, a classical two-state allosteric model best describes TetR function. Copyright © 2011 Elsevier Ltd. All rights reserved.

G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme.

PubMed

Hirschi, Alexander; Martin, William J; Luka, Zigmund; Loukachevitch, Lioudmila V; Reiter, Nicholas J

2016-08-01

Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono- and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG]8UUA) binds tightly to the functional LSD1-CoREST complex (Kd ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG]4U), a GQ DNA ([TTAGGG]4T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K(+)) is required for high affinity binding to the LSD1-CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms. © 2016 Hirschi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme

PubMed Central

Hirschi, Alexander; Martin, William J.; Luka, Zigmund; Loukachevitch, Lioudmila V.; Reiter, Nicholas J.

2016-01-01

Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono- and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG]8UUA) binds tightly to the functional LSD1–CoREST complex (Kd ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG]4U), a GQ DNA ([TTAGGG]4T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K+) is required for high affinity binding to the LSD1–CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms. PMID:27277658

HLA-A SNPs and amino acid variants are associated with nasopharyngeal carcinoma in Malaysian Chinese.

PubMed

Chin, Yoon-Ming; Mushiroda, Taisei; Takahashi, Atsushi; Kubo, Michiaki; Krishnan, Gopala; Yap, Lee-Fah; Teo, Soo-Hwang; Lim, Paul Vey-Hong; Yap, Yoke-Yeow; Pua, Kin-Choo; Kamatani, Naoyuki; Nakamura, Yusuke; Sam, Choon-Kook; Khoo, Alan Soo-Beng; Ng, Ching-Ching

2015-02-01

Nasopharyngeal carcinoma (NPC) arises from the mucosal epithelium of the nasopharynx and is constantly associated with Epstein-Barr virus type 1 (EBV-1) infection. We carried out a genome-wide association study (GWAS) of 575,247 autosomal SNPs in 184 NPC patients and 236 healthy controls of Malaysian Chinese ethnicity. Potential association signals were replicated in a separate cohort of 260 NPC patients and 245 healthy controls. We confirmed the association of HLA-A to NPC with the strongest signal detected in rs3869062 (p = 1.73 × 10(-9)). HLA-A fine mapping revealed associations in the amino acid variants as well as its corresponding SNPs in the antigen peptide binding groove (p(HLA-A-aa-site-99) = 3.79 × 10(-8), p(rs1136697) = 3.79 × 10(-8)) and T-cell receptor binding site (p(HLA-A-aa-site-145) = 1.41 × 10(-4), p(rs1059520) = 1.41 × 10(-4)) of the HLA-A. We also detected strong association signals in the 5'-UTR region with predicted active promoter states (p(rs41545520) = 7.91 × 10(-8)). SNP rs41545520 is a potential binding site for repressor ATF3, with increased binding affinity for rs41545520-G correlated with reduced HLA-A expression. Multivariate logistic regression diminished the effects of HLA-A amino acid variants and SNPs, indicating a correlation with the effects of HLA-A*11:01, and to a lesser extent HLA-A*02:07. We report the strong genetic influence of HLA-A on NPC susceptibility in the Malaysian Chinese. © 2014 UICC.

Direct Role for the Rpd3 Complex in Transcriptional Induction of the Anaerobic DAN/TIR Genes in Yeast▿‡

PubMed Central

Sertil, Odeniel; Vemula, Arvind; Salmon, Sharon L.; Morse, Randall H.; Lowry, Charles V.

2007-01-01

Saccharomyces cerevisiae adapts to hypoxia by expressing a large group of “anaerobic” genes. Among these, the eight DAN/TIR genes are regulated by the repressors Rox1 and Mot3 and the activator Upc2/Mox4. In attempting to identify factors recruited by the DNA binding repressor Mot3 to enhance repression of the DAN/TIR genes, we found that the histone deacetylase and global repressor complex, Rpd3-Sin3-Sap30, was not required for repression. Strikingly, the complex was instead required for activation. In addition, the histone H3 and H4 amino termini, which are targets of Rpd3, were also required for DAN1 expression. Epistasis tests demonstrated that the Rpd3 complex is not required in the absence of the repressor Mot3. Furthermore, the Rpd3 complex was required for normal function and stable binding of the activator Upc2 at the DAN1 promoter. Moreover, the Swi/Snf chromatin remodeling complex was strongly required for activation of DAN1, and chromatin immunoprecipitation analysis showed an Rpd3-dependent reduction in DAN1 promoter-associated nucleosomes upon induction. Taken together, these data provide evidence that during anaerobiosis, the Rpd3 complex acts at the DAN1 promoter to antagonize the chromatin-mediated repression caused by Mot3 and Rox1 and that chromatin remodeling by Swi/Snf is necessary for normal expression. PMID:17210643

Long Distance Modulation of Disorder-to-Order Transitions in Protein Allostery.

PubMed

Wang, Jingheng; Custer, Gregory; Beckett, Dorothy; Matysiak, Silvina

2017-08-29

Elucidation of the molecular details of allosteric communication between distant sites in a protein is key to understanding and manipulating many biological regulatory processes. Although protein disorder is acknowledged to play an important thermodynamic role in allostery, the molecular mechanisms by which this disorder is harnessed for long distance communication are known for a limited number of systems. Transcription repression by the Escherichia coli biotin repressor, BirA, is allosterically activated by binding of the small molecule effector biotinoyl-5'-AMP. The effector acts by promoting BirA dimerization, which is a prerequisite for sequence-specific binding to the biotin biosynthetic operon operator sequence. A 30 Å distance separates the effector binding and dimerization surfaces in BirA, and previous studies indicate that allostery is mediated, in part, by disorder-to-order transitions on the two coupled sites. In this work, combined experimental and computational methods have been applied to investigate the molecular basis of allosteric communication in BirA. Double-mutant cycle analysis coupled with thermodynamic measurements indicates functional coupling between residues in disordered loops on the two distant surfaces. All atom molecular dynamics simulations reveal that this coupling occurs through long distance reciprocal modulation of the structure and dynamics of disorder-to-order transitions on the two surfaces.

Dissociation free-energy profiles of specific and nonspecific DNA-protein complexes.

PubMed

Yonetani, Yoshiteru; Kono, Hidetoshi

2013-06-27

DNA-binding proteins recognize DNA sequences with at least two different binding modes: specific and nonspecific. Experimental structures of such complexes provide us a static view of the bindings. However, it is difficult to reveal further mechanisms of their target-site search and recognition only from static information because the transition process between the bound and unbound states is not clarified by static information. What is the difference between specific and nonspecific bindings? Here we performed adaptive biasing force molecular dynamics simulations with the specific and nonspecific structures of DNA-Lac repressor complexes to investigate the dissociation process. The resultant free-energy profiles showed that the specific complex has a sharp, deep well consistent with tight binding, whereas the nonspecific complex has a broad, shallow well consistent with loose binding. The difference in the well depth, ~5 kcal/mol, was in fair agreement with the experimentally obtained value and was found to mainly come from the protein conformational difference, particularly in the C-terminal tail. Also, the free-energy profiles were found to be correlated with changes in the number of protein-DNA contacts and that of surface water molecules. The derived protein spatial distributions around the DNA indicate that any large dissociation occurs rarely, regardless of the specific and nonspecific sites. Comparison of the free-energy barrier for sliding [~8.7 kcal/mol; Furini J. Phys. Chem. B 2010, 114, 2238] and that for dissociation (at least ~16 kcal/mol) calculated in this study suggests that sliding is much preferred to dissociation.

Molecular identification and transcriptional regulation of porcine IFIT2 gene.

PubMed

Yang, Xiuqin; Jing, Xiaoyan; Song, Yanfang; Zhang, Caixia; Liu, Di

2018-04-06

IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) plays important roles in host defense against viral infection as revealed by studies in humans and mice. However, little is known on porcine IFIT2 (pIFIT2). Here, we performed molecular cloning, expression profile, and transcriptional regulation analysis of pIFIT2. pIFIT2 gene, located on chromosome 14, is composed of two exons and have a complete coding sequence of 1407 bp. The encoded polypeptide, 468 aa in length, has three tetratricopeptide repeat motifs. pIFIT2 gene was unevenly distributed in all eleven tissues studied with the most abundance in spleen. Poly(I:C) treatment notably strongly upregulated the mRNA level and promoter activity of pIFIT2 gene. Upstream sequence of 1759 bp from the start codon which was assigned +1 here has promoter activity, and deltaEF1 acts as transcription repressor through binding to sequences at position - 1774 to - 1764. Minimal promoter region exists within nucleotide position - 162 and - 126. Two adjacent interferon-stimulated response elements (ISREs) and two nuclear factor (NF)-κB binding sites were identified within position - 310 and - 126. The ISRE elements act alone and in synergy with the one closer to start codon having more strength, so do the NF-κB binding sites. Synergistic effect was also found between the ISRE and NF-κB binding sites. Additionally, a third ISRE element was identified within position - 1661 to - 1579. These findings will contribute to clarifying the antiviral effect and underlying mechanisms of pIFIT2.

REST–Mediated Recruitment of Polycomb Repressor Complexes in Mammalian Cells

PubMed Central

Landt, Eskild; Agrawal-Singh, Shuchi; Bak, Mads; Tommerup, Niels; Rappsilber, Juri; Södersten, Erik; Hansen, Klaus

2012-01-01

Polycomb Repressive Complex (PRC) 1 and PRC2 regulate genes involved in differentiation and development. However, the mechanism for how PRC1 and PRC2 are recruited to genes in mammalian cells is unclear. Here we present evidence for an interaction between the transcription factor REST, PRC1, and PRC2 and show that RNF2 and REST co-regulate a number of neuronal genes in human teratocarcinoma cells (NT2-D1). Using NT2-D1 cells as a model of neuronal differentiation, we furthermore showed that retinoic-acid stimulation led to displacement of PRC1 at REST binding sites, reduced H3K27Me3, and increased gene expression. Genome-wide analysis of Polycomb binding in Rest−/− and Eed−/− mouse embryonic stem (mES) cells showed that Rest was required for PRC1 recruitment to a subset of Polycomb regulated neuronal genes. Furthermore, we found that PRC1 can be recruited to Rest binding sites independently of CpG islands and the H3K27Me3 mark. Surprisingly, PRC2 was frequently increased around Rest binding sites located in CpG-rich regions in the Rest−/− mES cells, indicating a more complex interplay where Rest also can limit PRC2 recruitment. Therefore, we propose that Rest has context-dependent functions for PRC1- and PRC2- recruitment, which allows this transcription factor to act both as a recruiter of Polycomb as well as a limiting factor for PRC2 recruitment at CpG islands. PMID:22396653

Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR

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

Carr, Carolyn E.; Musiani, Francesco; Huang, Hsin-Ting

Escherichia coli RcnR (resistance to cobalt and nickel regulator, EcRcnR) is a metal-responsive repressor of the genes encoding the Ni(II) and Co(II) exporter proteins RcnAB by binding to PRcnAB. The DNA binding affinity is weakened when the cognate ions Ni(II) and Co(II) bind to EcRcnR in a six-coordinate site that features a (N/O)5S ligand donor-atom set in distinct sites: while both metal ions are bound by the N terminus, Cys35, and His64, Co(II) is additionally bound by His3. On the other hand, the noncognate Zn(II) and Cu(I) ions feature a lower coordination number, have a solvent-accessible binding site, and coordinatemore » protein ligands that do not include the N-terminal amine. A molecular model of apo-EcRcnR suggested potential roles for Glu34 and Glu63 in binding Ni(II) and Co(II) to EcRcnR. The roles of Glu34 and Glu63 in metal binding, metal selectivity, and function were therefore investigated using a structure/function approach. X-ray absorption spectroscopy was used to assess the structural changes in the Ni(II), Co(II), and Zn(II) binding sites of Glu → Ala and Glu → Cys variants at both positions. The effect of these structural alterations on the regulation of PrcnA by EcRcnR in response to metal binding was explored using LacZ reporter assays. These combined studies indicate that while Glu63 is a ligand for both metal ions, Glu34 is a ligand for Co(II) but possibly not for Ni(II). The Glu34 variants affect the structure of the cognate metal sites, but they have no effect on the transcriptional response. In contrast, the Glu63 variants affect both the structure and transcriptional response, although they do not completely abolish the function of EcRcnR. The structure of the Zn(II) site is not significantly perturbed by any of the glutamic acid variations. The spectroscopic and functional data obtained on the mutants were used to calculate models of the metal-site structures of EcRcnR bound to Ni(II), Co(II), and Zn(II). The results are interpreted in terms of a switch mechanism, in which a subset of the metal-binding ligands is responsible for the allosteric response required for DNA release.« less

Bacillus subtilis 168 Contains Two Differentially Regulated Genes Encoding l-Asparaginase

PubMed Central

Fisher, Susan H.; Wray, Lewis V.

2002-01-01

Expression of the two Bacillus subtilis genes encoding l-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional l-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second l-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression. PMID:11914346

Bacillus subtilis 168 contains two differentially regulated genes encoding L-asparaginase.

PubMed

Fisher, Susan H; Wray, Lewis V

2002-04-01

Expression of the two Bacillus subtilis genes encoding L-asparaginase is controlled by independent regulatory factors. The ansZ gene (formerly yccC) was shown by mutational analysis to encode a functional L-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates ansZ expression by binding to a DNA site located upstream of the ansZ promoter. The expression of the ansA gene, which encodes the second L-asparaginase, was found to be induced by asparagine. The ansA repressor, AnsR, was shown to negatively regulate its own expression.

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

Engineered Single-Chain, Antiparallel, Coiled Coil Mimics the MerR Metal Binding Site

PubMed Central

Song, Lingyun; Caguiat, Jonathan; Li, Zhongrui; Shokes, Jacob; Scott, Robert A.; Olliff, Lynda; Summers, Anne O.

2004-01-01

The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of α-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue α-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR. In vivo, cells overexpressing MBD accumulated 70 to 100% more 203Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth. Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step. PMID:14996817

Viral Ubiquitin Ligase Stimulates Selective Host MicroRNA Expression by Targeting ZEB Transcriptional Repressors

PubMed Central

Kim, Ju Youn; Leader, Andrew; Stoller, Michelle L.; Coen, Donald M.; Wilson, Angus C.

2017-01-01

Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/δ-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors. PMID:28783105

THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

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

Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla

2011-01-07

Research highlights: {yields} THAP5 is a DNA-binding protein and a transcriptional repressor. {yields} THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. {yields} THAP5 induction correlates with the degree of apoptosis in melanoma cell population. {yields} THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown butmore » our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.« less

Mi2β Is Required for γ-Globin Gene Silencing: Temporal Assembly of a GATA-1-FOG-1-Mi2 Repressor Complex in β-YAC Transgenic Mice

PubMed Central

Costa, Flávia C.; Fedosyuk, Halyna; Chazelle, Allen M.; Neades, Renee Y.; Peterson, Kenneth R.

2012-01-01

Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the −566 GATA motif of the Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the −566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1–mediated repressor complex was disrupted by the −566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting −566 Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. PMID:23284307

Mi2β is required for γ-globin gene silencing: temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in β-YAC transgenic mice.

PubMed

Costa, Flávia C; Fedosyuk, Halyna; Chazelle, Allen M; Neades, Renee Y; Peterson, Kenneth R

2012-01-01

Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the -566 GATA motif of the (A)γ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of (A)γ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the -566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1-mediated repressor complex was disrupted by the -566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting -566 (A)γ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.

Engineering synthetic TAL effectors with orthogonal target sites

PubMed Central

Garg, Abhishek; Lohmueller, Jason J.; Silver, Pamela A.; Armel, Thomas Z.

2012-01-01

The ability to engineer biological circuits that process and respond to complex cellular signals has the potential to impact many areas of biology and medicine. Transcriptional activator-like effectors (TALEs) have emerged as an attractive component for engineering these circuits, as TALEs can be designed de novo to target a given DNA sequence. Currently, however, the use of TALEs is limited by degeneracy in the site-specific manner by which they recognize DNA. Here, we propose an algorithm to computationally address this problem. We apply our algorithm to design 180 TALEs targeting 20 bp cognate binding sites that are at least 3 nt mismatches away from all 20 bp sequences in putative 2 kb human promoter regions. We generated eight of these synthetic TALE activators and showed that each is able to activate transcription from a targeted reporter. Importantly, we show that these proteins do not activate synthetic reporters containing mismatches similar to those present in the genome nor a set of endogenous genes predicted to be the most likely targets in vivo. Finally, we generated and characterized TALE repressors comprised of our orthogonal DNA binding domains and further combined them with shRNAs to accomplish near complete repression of target gene expression. PMID:22581776

Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms.

PubMed

Xiao, Sheng; Yosef, Nir; Yang, Jianfei; Wang, Yonghui; Zhou, Ling; Zhu, Chen; Wu, Chuan; Baloglu, Erkan; Schmidt, Darby; Ramesh, Radha; Lobera, Mercedes; Sundrud, Mark S; Tsai, Pei-Yun; Xiang, Zhijun; Wang, Jinsong; Xu, Yan; Lin, Xichen; Kretschmer, Karsten; Rahl, Peter B; Young, Richard A; Zhong, Zhong; Hafler, David A; Regev, Aviv; Ghosh, Shomir; Marson, Alexander; Kuchroo, Vijay K

2014-04-17

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity. Copyright © 2014 Elsevier Inc. All rights reserved.

Protein-mediated loops in supercoiled DNA create large topological domains

PubMed Central

Yan, Yan; Ding, Yue; Leng, Fenfei; Dunlap, David; Finzi, Laura

2018-01-01

Abstract Supercoiling can alter the form and base pairing of the double helix and directly impact protein binding. More indirectly, changes in protein binding and the stress of supercoiling also influence the thermodynamic stability of regulatory, protein-mediated loops and shift the equilibria of fundamental DNA/chromatin transactions. For example, supercoiling affects the hierarchical organization and function of chromatin in topologically associating domains (TADs) in both eukaryotes and bacteria. On the other hand, a protein-mediated loop in DNA can constrain supercoiling within a plectonemic structure. To characterize the extent of constrained supercoiling, 400 bp, lac repressor-secured loops were formed in extensively over- or under-wound DNA under gentle tension in a magnetic tweezer. The protein-mediated loops constrained variable amounts of supercoiling that often exceeded the maximum writhe expected for a 400 bp plectoneme. Loops with such high levels of supercoiling appear to be entangled with flanking domains. Thus, loop-mediating proteins operating on supercoiled substrates can establish topological domains that may coordinate gene regulation and other DNA transactions across spans in the genome that are larger than the separation between the binding sites. PMID:29538766

Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

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

Pazehoski, Kristina O., E-mail: pazehosk@pitt.edu; Cobine, Paul A., E-mail: pac0006@auburn.edu; Winzor, Donald J.

2011-03-11

Research highlights: {yields} A metal-binding protein domain is directly involved in protein dimerization. {yields} Fusing the metal-binding domain to a monomeric protein induces dimerization. {yields} Frontal size-exclusion chromatography measures the strength of dimer interaction. {yields} Ultracentrifugation studies confirm the influence of metal binding on dimerization. -- Abstract: Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size-exclusion chromatography studies. The studies demonstrate thatmore » the attachment of these CopY residues to the immunoglobulin-binding domain of streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.« less

Insights into GATA-1 Mediated Gene Activation versus Repression via Genome-wide Chromatin Occupancy Analysis

PubMed Central

Yu, Ming; Riva, Laura; Xie, Huafeng; Schindler, Yocheved; Moran, Tyler B.; Cheng, Yong; Yu, Duonan; Hardison, Ross; Weiss, Mitchell J; Orkin, Stuart H.; Bernstein, Bradley E.; Fraenkel, Ernest; Cantor, Alan B.

2009-01-01

Summary The transcription factor GATA-1 is required for terminal erythroid maturation and functions as an activator or repressor depending on gene context. Yet its in vivo site selectivity and ability to distinguish between activated versus repressed genes remain incompletely understood. In this study, we performed GATA-1 ChIP-seq in erythroid cells and compared it to GATA-1 induced gene expression changes. Bound and differentially expressed genes contain a greater number of GATA binding motifs, a higher frequency of palindromic GATA sites, and closer occupancy to the transcriptional start site versus non-differentially expressed genes. Moreover, we show that the transcription factor Zbtb7a occupies GATA-1 bound regions of some direct GATA-1 target genes, that the presence of SCL/TAL1 helps distinguish transcriptional activation versus repression, and that Polycomb Repressive Complex 2 (PRC2) is involved in epigenetic silencing of a subset of GATA-1 repressed genes. These data provide insights into GATA-1 mediated gene regulation in vivo. PMID:19941827

FY is an RNA 3' end-processing factor that interacts with FCA to control the Arabidopsis floral transition.

PubMed

Simpson, Gordon G; Dijkwel, Paul P; Quesada, Victor; Henderson, Ian; Dean, Caroline

2003-06-13

The nuclear RNA binding protein, FCA, promotes Arabidopsis reproductive development. FCA contains a WW protein interaction domain that is essential for FCA function. We have identified FY as a protein partner for this domain. FY belongs to a highly conserved group of eukaryotic proteins represented in Saccharomyces cerevisiae by the RNA 3' end-processing factor, Pfs2p. FY regulates RNA 3' end processing in Arabidopsis as evidenced through its role in FCA regulation. FCA expression is autoregulated through the use of different polyadenylation sites within the FCA pre-mRNA, and the FCA/FY interaction is required for efficient selection of the promoter-proximal polyadenylation site. The FCA/FY interaction is also required for the downregulation of the floral repressor FLC. We propose that FCA controls 3' end formation of specific transcripts and that in higher eukaryotes, proteins homologous to FY may have evolved as sites of association for regulators of RNA 3' end processing.

Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network.

PubMed

Culyba, Matthew J; Kubiak, Jeffrey M; Mo, Charlie Y; Goulian, Mark; Kohli, Rahul M

2018-06-01

Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter's activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of 'early', 'middle', and 'late' genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene's transcription as a function of stimulus dose.

Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition.

PubMed

Duss, Olivier; Michel, Erich; Diarra dit Konté, Nana; Schubert, Mario; Allain, Frédéric H-T

2014-04-01

The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.

Efficient analysis of stochastic gene dynamics in the non-adiabatic regime using piecewise deterministic Markov processes

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

Lin, Yen Ting; Buchler, Nicolas E.

Single-cell experiments show that gene expression is stochastic and bursty, a feature that can emerge from slow switching between promoter states with different activities. In addition to slow chromatin and/or DNA looping dynamics, one source of long-lived promoter states is the slow binding and unbinding kinetics of transcription factors to promoters, i.e. the non-adiabatic binding regime. Here, we introduce a simple analytical framework, known as a piecewise deterministic Markov process (PDMP), that accurately describes the stochastic dynamics of gene expression in the non-adiabatic regime. We illustrate the utility of the PDMP on a non-trivial dynamical system by analysing the propertiesmore » of a titration-based oscillator in the non-adiabatic limit. We first show how to transform the underlying chemical master equation into a PDMP where the slow transitions between promoter states are stochastic, but whose rates depend upon the faster deterministic dynamics of the transcription factors regulated by these promoters. We show that the PDMP accurately describes the observed periods of stochastic cycles in activator and repressor-based titration oscillators. We then generalize our PDMP analysis to more complicated versions of titration-based oscillators to explain how multiple binding sites lengthen the period and improve coherence. Finally, we show how noise-induced oscillation previously observed in a titration-based oscillator arises from non-adiabatic and discrete binding events at the promoter site.« less

Induction of dystrophin Dp71 expression during neuronal differentiation: opposite roles of Sp1 and AP2alpha in Dp71 promoter activity.

PubMed

Morales-Lázaro, Sara Luz; González-Ramírez, Ricardo; Gómez, Pablo; Tapia-Ramírez, Victor; de León, Mario Bermúdez; Cisneros, Bulmaro

2010-01-01

In this study, we delineated the molecular mechanisms that modulate Dp71 expression during neuronal differentiation, using the N1E-115 cell line. We demonstrated that Dp71 expression is up-regulated in response to cAMP-mediated neuronal differentiation of these cells, and that this induction is controlled at promoter level. Functional deletion analysis of the Dp71 promoter revealed that a 5'-flanking 159-bp DNA fragment that contains Sp1 and AP2 binding sites is necessary and sufficient for basal expression of this TATA-less promoter, as well as for its induction during neuronal differentiation. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that Sp1 and AP2alpha bind to their respective DNA elements within the Dp71 basal promoter. Overall, mutagenesis assays on the Sp1 and AP2 binding sites, over-expression of Sp1 and AP2alpha, as well as knock-down experiments on Sp1 and AP2alpha gene expression established that Dp71 basal expression is controlled by the combined action of Sp1 and AP2alpha, which act as activator and repressor, respectively. Furthermore, we demonstrated that induction of Dp71 expression in differentiated cells is the result of the maintenance of positive regulation exerted by Sp1, as well as of the loss of AP2alpha binding, which ultimately releases the promoter from repression.

Tannic acid facilitates expression of the polypyrimidine tract binding protein and alleviates deleterious inclusion of CHRNA1 exon P3A due to an hnRNP H-disrupting mutation in congenital myasthenic syndrome

PubMed Central

Bian, Yang; Masuda, Akio; Matsuura, Tohru; Ito, Mikako; Okushin, Kazuya; Engel, Andrew G.; Ohno, Kinji

2009-01-01

We recently reported that the intronic splice-site mutation IVS3-8G>A of CHRNA1 that encodes the muscle nicotinic acetylcholine receptor α subunit disrupts binding of a splicing repressor, hnRNP H. This, in turn, results in exclusive inclusion of the downstream exon P3A. The P3A(+) transcript encodes a non-functional α subunit that comprises 50% of the transcripts in normal human skeletal muscle, but its functional significance remains undetermined. In an effort to search for a potential therapy, we screened off-label effects of 960 bioactive chemical compounds and found that tannic acid ameliorates the aberrant splicing due to IVS3-8G>A but without altering the expression of hnRNP H. Therefore, we searched for another splicing trans-factor. We found that the polypyrimidine tract binding protein (PTB) binds close to the 3′ end of CHRNA1 intron 3, that PTB induces skipping of exon P3A and that tannic acid increases the expression of PTB in a dose-dependent manner. Deletion assays of the PTB promoter region revealed that the tannic acid-responsive element is between positions −232 and −74 from the translation initiation site. These observations open the door to the discovery of novel therapies based on PTB overexpression and to detecting possible untoward effects of the overexpression. PMID:19147685

Efficient analysis of stochastic gene dynamics in the non-adiabatic regime using piecewise deterministic Markov processes

DOE PAGES

Lin, Yen Ting; Buchler, Nicolas E.

2018-01-31

Single-cell experiments show that gene expression is stochastic and bursty, a feature that can emerge from slow switching between promoter states with different activities. In addition to slow chromatin and/or DNA looping dynamics, one source of long-lived promoter states is the slow binding and unbinding kinetics of transcription factors to promoters, i.e. the non-adiabatic binding regime. Here, we introduce a simple analytical framework, known as a piecewise deterministic Markov process (PDMP), that accurately describes the stochastic dynamics of gene expression in the non-adiabatic regime. We illustrate the utility of the PDMP on a non-trivial dynamical system by analysing the propertiesmore » of a titration-based oscillator in the non-adiabatic limit. We first show how to transform the underlying chemical master equation into a PDMP where the slow transitions between promoter states are stochastic, but whose rates depend upon the faster deterministic dynamics of the transcription factors regulated by these promoters. We show that the PDMP accurately describes the observed periods of stochastic cycles in activator and repressor-based titration oscillators. We then generalize our PDMP analysis to more complicated versions of titration-based oscillators to explain how multiple binding sites lengthen the period and improve coherence. Finally, we show how noise-induced oscillation previously observed in a titration-based oscillator arises from non-adiabatic and discrete binding events at the promoter site.« less

In silico modeling of epigenetic-induced changes in photoreceptor cis-regulatory elements.

PubMed

Hossain, Reafa A; Dunham, Nicholas R; Enke, Raymond A; Berndsen, Christopher E

2018-01-01

DNA methylation is a well-characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of transcription is controlled by proteins that regulate recruitment and activity of RNA polymerase by binding to specific cis-regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell-specific gene expression. Although much is known about the functions and DNA binding specificity of CRX, little is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements. We used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO , PDE6B, PAX6 , and LINE1 retrotransposon repeats. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences. In this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to the DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate changes in the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX-dependent transcription. Collectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

Transcription factor clusters regulate genes in eukaryotic cells

PubMed Central

Hedlund, Erik G; Friemann, Rosmarie; Hohmann, Stefan

2017-01-01

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression. PMID:28841133

Genome-wide survey by ChIP-seq reveals YY1 regulation of lincRNAs in skeletal myogenesis

PubMed Central

Lu, Leina; Sun, Kun; Chen, Xiaona; Zhao, Yu; Wang, Lijun; Zhou, Liang; Sun, Hao; Wang, Huating

2013-01-01

Skeletal muscle differentiation is orchestrated by a network of transcription factors, epigenetic regulators, and non-coding RNAs. The transcription factor Yin Yang 1 (YY1) silences multiple target genes in myoblasts (MBs) by recruiting Ezh2 (Enhancer of Zeste Homologue2). To elucidate genome-wide YY1 binding in MBs, we performed chromatin immunoprecipitation (ChIP)-seq and found 1820 specific binding sites in MBs with a large portion residing in intergenic regions. Detailed analysis demonstrated that YY1 acts as an activator for many loci in addition to its known repressor function. No significant co-occupancy was found between YY1 and Ezh2, suggesting an additional Ezh2-independent function for YY1 in MBs. Further analysis of intergenic binding sites showed that YY1 potentially regulates dozens of large intergenic non-coding RNAs (lincRNAs), whose function in myogenesis is underexplored. We characterized a novel muscle-associated lincRNA (Yam-1) that is positively regulated by YY1. Yam-1 is downregulated upon differentiation and acts as an inhibitor of myogenesis. We demonstrated that Yam-1 functions through in cis regulation of miR-715, which in turn targets Wnt7b. Our findings not only provide the first genome-wide picture of YY1 association in muscle cells, but also uncover the functional role of lincRNA Yam-1. PMID:23942234

Functional domains of the human splicing factor ASF/SF2.

PubMed Central

Zuo, P; Manley, J L

1993-01-01

The human splicing factor ASF/SF2 displays two predominant activities in in vitro splicing assays: (i) it is an essential factor apparently required for all splices and (ii) it is able to switch utilization of alternative 5' splice sites in a concentration-dependent manner. ASF/SF2 is the prototype of a family of proteins typified by the presence of one or two RNP-type RNA binding domains (RBDs) and a region highly enriched in repeating arginine-serine dipeptides (RS regions). Here we describe a functional analysis of ASF/SF2, which defines several regions essential for one, or both, of its two principal activities, and provides insights into how this type of protein functions in splicing. Two isoforms of the protein, which arise from alternative splicing, are by themselves inactive, but each can block the activity of ASF/SF2, thereby functioning as splicing repressors. Some, but not all, mutations in the RS region prevent ASF/SF2 from functioning as an essential splicing factor. However, the entire RS region can be deleted without reducing splice site switching activity, indicating that it is not absolutely required for interaction with other splicing factors. Experiments with deletion and substitution mutants reveal that the protein contains two related, but highly diverged, RBDs, and that both are essential for activity. Each RBD by itself retains the ability to bind RNA, although optimal binding requires both domains. Images PMID:8223481

Effects of atorvastatin metabolites on induction of drug-metabolizing enzymes and membrane transporters through human pregnane X receptor

PubMed Central

Hoffart, E; Ghebreghiorghis, L; Nussler, AK; Thasler, WE; Weiss, TS; Schwab, M; Burk, O

2012-01-01

BACKGROUND AND PURPOSE Atorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). EXPERIMENTAL APPROACH Ligand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation. KEY RESULTS All atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors. CONCLUSIONS AND IMPLICATIONS Atorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound. PMID:21913896

Disruption of DNA methylation-dependent long gene repression in Rett syndrome

PubMed Central

Gabel, Harrison W.; Kinde, Benyam Z.; Stroud, Hume; Gilbert, Caitlin S.; Harmin, David A.; Kastan, Nathaniel R.; Hemberg, Martin; Ebert, Daniel H.; Greenberg, Michael E.

2015-01-01

Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism1. MECP2 encodes a methyl-DNA-binding protein2 that has been proposed to function as a transcriptional repressor, but despite numerous studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 regulates transcription3–9. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain. PMID:25762136

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

PubMed

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

2014-12-20

Germlines in plants are formed de novo during post-embryonic development, while little is known about the mechanism that controls this process. In Arabidopsis, the earliest gene controlling this process is SPOROCYTELESS (SPL). A decade ago, we showed that loss of SPL function abolished sporogenesis in both male and female organs of Arabidopsis. However, its function is unclear up to now. In this study, we showed that SPL belongs to a novel transcription repressor family specific in embryophyte, which consists of 173 members in the land plants so far. All of them contain a conserved SPL-motif in their N-terminal and an ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif in the C-terminal, therefore designated as SPL-like, EAR-containing proteins (SPEARs). Consistently, SPL acts as a transcriptional repressor in yeast and tobacco cells, and SPEAR proteins are able to form homodimer and/or heterodimer with each other in vitro. Furthermore, SPEARs interact with the TOPLESS (TPL) co-repressors via the EAR motif and TCP family transcription factors in yeast cells. Together, we propose that SPL and SPEARs most likely belong to a novel transcription repressor family in land plants which may play a variety of developmental roles in plants. Copyright © 2014 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression.

PubMed

van Rooijen, R J; Dechering, K J; Niek, C; Wilmink, J; de Vos, W M

1993-02-01

Site-directed mutagenesis of the Lactococcus lactis lacR gene was performed to identify residues in the LacR repressor that are involved in the induction of lacABCDFEGX operon expression by tagatose-6-phosphate. A putative inducer binding domain located near the C-terminus was previously postulated based on homology studies with the Escherichia coli DeoR family of repressors, which all have a phosphorylated sugar as inducer. Residues within this domain and lysine residues that are charge conserved in the DeoR family were changed into alanine or arginine. The production of the LacR mutants K72A, K80A, K80R, D210A, K213A and K213R in the LacR-deficient L.lactis strain NZ3015 resulted in repressed phospho-beta-galactosidase (LacG) activities and decreased growth rates on lactose. Gel mobility shift assays showed that the complex between a DNA fragment carrying the lac operators and LacR mutants K72A, K80A, K213A and D210A did not dissociate in the presence of tagatose-6-phosphate, in contrast to wild type LacR. Other mutations (K62A/K63A, K72R, K73A, K73R, T212A, F214R, R216R and R216K) exhibited no gross effects on inducer response. The results strongly suggest that the lysines at positions 72, 80 and 213 and aspartic acid at position 210 are involved in the induction of lac operon expression by tagatose-6-phosphate.

R1, a novel repressor of the human monoamine oxidase A.

PubMed

Chen, Kevin; Ou, Xiao-Ming; Chen, Gao; Choi, Si Ho; Shih, Jean C

2005-03-25

Monoamine oxidase catalyzes the oxidative deamination of a number of neurotransmitters. A deficiency in monoamine oxidase A results in aggressive behavior in both humans and mice. Studies on the regulation of monoamine oxidase A gene expression have shown that the Sp1 family is important for monoamine oxidase A expression. To search for novel transcription factors, the sequences of three Sp1 sites in the monoamine oxidase A core promoter were used in the yeast one-hybrid system to screen a human cDNA library. A novel repressor, R1 (RAM2), has been cloned. The R1 cDNA encodes a protein with 454 amino acids and an open reading frame at the 5'-end. The transfection of R1 in a human neuroblastoma cell line, SK-N-BE (2)-C, inhibited the monoamine oxidase A promoter and enzymatic activity. The degree of inhibition of monoamine oxidase A by R1 correlated with the level of R1 protein expression. R1 was also found to repress monoamine oxidase A promoter activity within a natural chromatin environment. A gel-shift assay indicated that the endogenous R1 protein in SK-N-BE (2)-C cells interacted with the R1 binding sequence. R1 also bound directly to the natural monoamine oxidase A promoter in vivo as shown by chromatin immunoprecipitation assay. Immunocytochemical analysis showed that R1 was expressed in both cytosol and nucleus, which suggested a role for R1 in transcriptional regulation. Northern blot analysis revealed the presence of endogenous R1 mRNA in human brain and peripheral tissues. Taken together, this study shows that R1 is a novel repressor that inhibits monoamine oxidase A gene expression.

A novel corepressor, BCoR-L1, represses transcription through an interaction with CtBP.

PubMed

Pagan, Julia K; Arnold, Jeremy; Hanchard, Kim J; Kumar, Raman; Bruno, Tiziana; Jones, Mathew J K; Richard, Derek J; Forrest, Alistair; Spurdle, Amanda; Verdin, Eric; Crossley, Merlin; Fanciulli, Maurizio; Chenevix-Trench, Georgia; Young, David B; Khanna, Kum Kum

2007-05-18

Corepressors play a crucial role in negative gene regulation and are defective in several diseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe and functionally characterize BCoR-L1, a homolog of BCoR. When tethered to a heterologous promoter, BCoR-L1 is capable of strong repression. Like other corepressors, BCoR-L1 associates with histone deacetylase (HDAC) activity. Specifically, BCoR-L1 coprecipitates with the Class II HDACs, HDAC4, HDAC5, and HDAC7, suggesting that they are involved in its role as a transcriptional repressor. BCoR-L1 also interacts with the CtBP corepressor through a CtBP-interacting motif in its amino terminus. Abrogation of the CtBP binding site within BCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore, BCoR-L1 is located on the E-cadherin promoter, a known CtBP-regulated promoter, and represses the E-cadherin promoter activity in a reporter assay. The inhibition of BCoR-L1 expression by RNA-mediated interference results in derepression of E-cadherin in cells that do not normally express E-cadherin, indicating that BCoR-L1 contributes to the repression of an authentic endogenous CtBP target.

Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping

PubMed Central

Normanno, Davide; Vanzi, Francesco; Pavone, Francesco Saverio

2008-01-01

Gene expression regulation is a fundamental biological process which deploys specific sets of genomic information depending on physiological or environmental conditions. Several transcription factors (including lac repressor, LacI) are present in the cell at very low copy number and increase their local concentration by binding to multiple sites on DNA and looping the intervening sequence. In this work, we employ single-molecule manipulation to experimentally address the role of DNA supercoiling in the dynamics and stability of LacI-mediated DNA looping. We performed measurements over a range of degrees of supercoiling between −0.026 and +0.026, in the absence of axial stretching forces. A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed. Our experiments also provide evidence for multiple structural conformations of the LacI–DNA complex, depending on torsional constraints. The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon. In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes. PMID:18310101

Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches

PubMed Central

Hoffmann, Stefan A.; Kruse, Sabrina M.; Arndt, Katja M.

2016-01-01

Abstract We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ70 dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a ‘forward’ gene interferes with the expression of a ‘reverse’ gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection. PMID:26932362

Structure of the apo form of the catabolite control protein A (CcpA) from Bacillus megaterium with a DNA-binding domain

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

Singh, Rajesh Kumar; Palm, Gottfried J.; Panjikar, Santosh

2007-04-01

Crystal structure analysis of the apo form of catabolite control protein A reveals the three-helix bundle of the DNA-binding domain. In the crystal packing, this domain interacts with the binding site for the corepressor protein. Crystal structure determination of catabolite control protein A (CcpA) at 2.6 Å resolution reveals for the first time the structure of a full-length apo-form LacI-GalR family repressor protein. In the crystal structures of these transcription regulators, the three-helix bundle of the DNA-binding domain has only been observed in cognate DNA complexes; it has not been observed in other crystal structures owing to its mobility. Inmore » the crystal packing of apo-CcpA, the protein–protein contacts between the N-terminal three-helix bundle and the core domain consisted of interactions between the homodimers that were similar to those between the corepressor protein HPr and the CcpA N-subdomain in the ternary DNA complex. In contrast to the DNA complex, the apo-CcpA structure reveals large subdomain movements in the core, resulting in a complete loss of contacts between the N-subdomains of the homodimer.« less

RARα-PLZF overcomes PLZF-mediated repression of CRABPI, contributing to retinoid resistance in t(11;17) acute promyelocytic leukemia

PubMed Central

Guidez, Fabien; Parks, Sarah; Wong, Henna; Jovanovic, Jelena V.; Mays, Ashley; Gilkes, Amanda F.; Mills, Kenneth I.; Guillemin, Marie-Claude; Hobbs, Robin M.; Pandolfi, Pier Paolo; de Thé, Hugues; Solomon, Ellen; Grimwade, David

2007-01-01

Leukemia-associated chimeric oncoproteins often act as transcriptional repressors, targeting promoters of master genes involved in hematopoiesis. We show that CRABPI (encoding cellular retinoic acid binding protein I) is a target of PLZF, which is fused to RARα by the t(11;17)(q23;q21) translocation associated with retinoic acid (RA)-resistant acute promyelocytic leukemia (APL). PLZF represses the CRABPI locus through propagation of chromatin condensation from a remote intronic binding element culminating in silencing of the promoter. Although the canonical, PLZF-RARα oncoprotein has no impact on PLZF-mediated repression, the reciprocal translocation product RARα-PLZF binds to this remote binding site, recruiting p300, inducing promoter hypomethylation and CRABPI gene up-regulation. In line with these observations, RA-resistant murine PLZF/RARα+RARα/PLZF APL blasts express much higher levels of CRABPI than standard RA-sensitive PML/RARα APL. RARα-PLZF confers RA resistance to a retinoid-sensitive acute myeloid leukemia (AML) cell line in a CRABPI-dependent fashion. This study supports an active role for PLZF and RARα-PLZF in leukemogenesis, identifies up-regulation of CRABPI as a mechanism contributing to retinoid resistance, and reveals the ability of the reciprocal fusion gene products to mediate distinct epigenetic effects contributing to the leukemic phenotype. PMID:18000064

Combined cistrome and transcriptome analysis of SKI in AML cells identifies SKI as a co-repressor for RUNX1

PubMed Central

Feld, Christine; Sahu, Peeyush; Frech, Miriam; Finkernagel, Florian; Nist, Andrea; Stiewe, Thorsten; Bauer, Uta-Maria; Neubauer, Andreas

2018-01-01

Abstract SKI is a transcriptional co-regulator and overexpressed in various human tumors, for example in acute myeloid leukemia (AML). SKI contributes to the origin and maintenance of the leukemic phenotype. Here, we use ChIP-seq and RNA-seq analysis to identify the epigenetic alterations induced by SKI overexpression in AML cells. We show that approximately two thirds of differentially expressed genes are up-regulated upon SKI deletion, of which >40% harbor SKI binding sites in their proximity, primarily in enhancer regions. Gene ontology analysis reveals that many of the differentially expressed genes are annotated to hematopoietic cell differentiation and inflammatory response, corroborating our finding that SKI contributes to a myeloid differentiation block in HL60 cells. We find that SKI peaks are enriched for RUNX1 consensus motifs, particularly in up-regulated SKI targets upon SKI deletion. RUNX1 ChIP-seq displays that nearly 70% of RUNX1 binding sites overlap with SKI peaks, mainly at enhancer regions. SKI and RUNX1 occupy the same genomic sites and cooperate in gene silencing. Our work demonstrates for the first time the predominant co-repressive function of SKI in AML cells on a genome-wide scale and uncovers the transcription factor RUNX1 as an important mediator of SKI-dependent transcriptional repression. PMID:29471413

Bombyx mori E26 transformation-specific 2 (BmEts2), an Ets family protein, represses Bombyx mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in the silkworm Bombyx mori.

PubMed

Tanaka, H; Sagisaka, A; Suzuki, N; Yamakawa, M

2016-10-01

E26 transformation-specific (Ets) family transcription factors are known to play roles in various biological phenomena, including immunity, in vertebrates. However, the mechanisms by which Ets proteins contribute to immunity in invertebrates remain poorly understood. In this study, we identified a cDNA encoding BmEts2, which is a putative orthologue of Drosophila Yan and human translocation-ets-leukemia/Ets-variant gene 6, from the silkworm Bombyx mori. Expression of the BmEts2 gene was significantly increased in the fat bodies of silkworm larvae in response to injection with Escherichia coli and Staphylococcus aureus. BmEts2 overexpression dramatically repressed B. mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in silkworm cells. Conversely, gene knockdown of BmEts2 significantly enhanced BmRels activity. In addition, two κB sites located on the 5' upstream region of cecropin B1 were found to be involved in the repression of BmRels-mediated promoter activation. Protein-competition analysis further demonstrated that BmEts2 competitively inhibited binding of BmRels to κB sites. Overall, BmEts2 acts as a repressor of BmRels-mediated transactivation of antimicrobial protein genes by inhibiting the binding of BmRels to κB sites. © 2016 The Royal Entomological Society.

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

PubMed

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

2017-04-01

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

Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation.

PubMed

Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G; Xie, Jiuyong

2012-09-01

The molecular basis of cell signal-regulated alternative splicing at the 3' splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3' splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3' splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3' splice site usage.

The activity of CouR, a MarR family transcriptional regulator, is modulated through a novel molecular mechanism

DOE PAGES

Otani, Hiroshi; Stogios, Peter J.; Xu, Xiaohui; ...

2015-09-22

CouR, a MarR-type transcriptional repressor, regulates the cou genes, encoding p-hydroxycinnamate catabolism in the soil bacterium Rhodococcus jostii RHA1. The CouR dimer bound two molecules of the catabolite p-coumaroyl–CoA (K d = 11 ± 1 μM). The presence of p-coumaroyl–CoA, but neither p-coumarate nor CoASH, abrogated CouR's binding to its operator DNA in vitro. The crystal structures of ligand-free CouR and its p-coumaroyl–CoA-bound form showed no significant conformational differences, in contrast to other MarR regulators. The CouR– p-coumaroyl–CoA structure revealed two ligand molecules bound to the CouR dimer with their phenolic moieties occupying equivalent hydrophobic pockets in each protomer andmore » their CoA moieties adopting non-equivalent positions to mask the regulator's predicted DNA-binding surface. More specifically, the CoA phosphates formed salt bridges with predicted DNA-binding residues Arg36 and Arg38, changing the overall charge of the DNA-binding surface. The substitution of either arginine with alanine completely abrogated the ability of CouR to bind DNA. By contrast, the R36A/R38A double variant retained a relatively high affinity for p-coumaroyl–CoA (K d = 89 ± 6 μM). Altogether, our data point to a novel mechanism of action in which the ligand abrogates the repressor's ability to bind DNA by steric occlusion of key DNA-binding residues and charge repulsion of the DNA backbone.« less

Molecular basis for repression of liver X receptor-mediated gene transcription by receptor-interacting protein 140

PubMed Central

Jakobsson, Tomas; Osman, Waffa; Gustafsson, Jan-Åke; Zilliacus, Johanna; Wärnmark, Anette

2007-01-01

Similarities in physiological roles of LXR (liver X receptors) and co-repressor RIP140 (receptor-interacting protein 140) in regulating energy homoeostasis and lipid and glucose metabolism suggest that the effects of LXR could at least partly be mediated by recruitment of the co-repressor RIP140. In the present study, we have elucidated the molecular basis for regulation of LXR transcriptional activity by RIP140. LXR is evenly localized in the nucleus and neither the N-terminal domain nor the LBD (ligand-binding domain) is necessary for nuclear localization. Both LXR subtypes, LXRα and LXRβ, interact with RIP140 and co-localize in diffuse large nuclear domains. Interaction and co-localization are dependent on the LBD of the receptor. The C-terminal domain of RIP140 is sufficient for full repressive effect. None of the C-terminal NR (nuclear receptor)-boxes is required for the co-repressor activity, whereas the NR-box-like motif as well as additional elements in the C-terminal region are required for full repressive function. The C-terminal NR-box-like motif is necessary for interaction with LXRβ, whereas additional elements are needed for strong interaction with LXRα. In conclusion, our results suggest that co-repression of LXR activity by RIP140 involves an atypical binding mode of RIP140 and a repression element in the RIP140 C-terminus. PMID:17391100

Animal-specific C-terminal domain links myeloblastosis oncoprotein (Myb) to an ancient repressor complex

PubMed Central

Andrejka, Laura; Wen, Hong; Ashton, Jonathan; Grant, Megan; Iori, Kevin; Wang, Amy; Manak, J. Robert; Lipsick, Joseph S.

2011-01-01

Members of the Myb oncoprotein and E2F-Rb tumor suppressor protein families are present within the same highly conserved multiprotein transcriptional repressor complex, named either as Myb and synthetic multivuval class B (Myb-MuvB) or as Drosophila Rb E2F and Myb-interacting proteins (dREAM). We now report that the animal-specific C terminus of Drosophila Myb but not the more highly conserved N-terminal DNA-binding domain is necessary and sufficient for (i) adult viability, (ii) proper localization to chromosomes in vivo, (iii) regulation of gene expression in vivo, and (iv) interaction with the highly conserved core of the MuvB/dREAM transcriptional repressor complex. In addition, we have identified a conserved peptide motif that is required for this interaction. Our results imply that an ancient function of Myb in regulating G2/M genes in both plants and animals appears to have been transferred from the DNA-binding domain to the animal-specific C-terminal domain. Increased expression of B-MYB/MYBL2, the human ortholog of Drosophila Myb, correlates with poor prognosis in human patients with breast cancer. Therefore, our results imply that the specific interaction of the C terminus of Myb with the MuvB/dREAM core complex may provide an attractive target for the development of cancer therapeutics. PMID:21969598

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila

PubMed Central

Ji, Yingbiao

2016-01-01

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates the posttranscriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germ line stem cell maintenance and oocyte localization. The mRNA targets regulated by Hrp38 are mostly unknown. We identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. We found that Hrp38 binds to the 3′ untranslated region (UTR) of Nanos mRNA, which contains a translation control element. We have demonstrated that translation of the luciferase reporter bearing the Nanos 3′ UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. Our data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to the Nanos 3′ UTR, increasing the translation in vivo and in vitro. hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. We conclude that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis. PMID:27402862

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

PubMed

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

2000-11-10

In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells. Copyright 2000 Academic Press.

A Simple Negative Interaction in the Positive Transcriptional Feedback of a Single Gene Is Sufficient to Produce Reliable Oscillations

PubMed Central

Miró-Bueno, Jesús M.; Rodríguez-Patón, Alfonso

2011-01-01

Negative and positive transcriptional feedback loops are present in natural and synthetic genetic oscillators. A single gene with negative transcriptional feedback needs a time delay and sufficiently strong nonlinearity in the transmission of the feedback signal in order to produce biochemical rhythms. A single gene with only positive transcriptional feedback does not produce oscillations. Here, we demonstrate that this single-gene network in conjunction with a simple negative interaction can also easily produce rhythms. We examine a model comprised of two well-differentiated parts. The first is a positive feedback created by a protein that binds to the promoter of its own gene and activates the transcription. The second is a negative interaction in which a repressor molecule prevents this protein from binding to its promoter. A stochastic study shows that the system is robust to noise. A deterministic study identifies that the dynamics of the oscillator are mainly driven by two types of biomolecules: the protein, and the complex formed by the repressor and this protein. The main conclusion of this paper is that a simple and usual negative interaction, such as degradation, sequestration or inhibition, acting on the positive transcriptional feedback of a single gene is a sufficient condition to produce reliable oscillations. One gene is enough and the positive transcriptional feedback signal does not need to activate a second repressor gene. This means that at the genetic level an explicit negative feedback loop is not necessary. The model needs neither cooperative binding reactions nor the formation of protein multimers. Therefore, our findings could help to clarify the design principles of cellular clocks and constitute a new efficient tool for engineering synthetic genetic oscillators. PMID:22205920

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. Copyright 2004 Wiley Periodicals, Inc.

Transcriptional co-repressor SIN3A silencing rescues decline in memory consolidation during scopolamine-induced amnesia.

PubMed

Srivas, Sweta; Thakur, Mahendra K

2018-05-01

Epigenetic modifications through methylation of DNA and acetylation of histones modulate neuronal gene expression and regulate long-term memory. Earlier we demonstrated that scopolamine-induced decrease in memory consolidation is correlated with enhanced expression of hippocampal DNA methyltransferase 1 (DNMT1) and histone deacetylase 2 (HDAC2) in mice. DNMT1 and HDAC2 act together by recruiting a co-repressor complex and deacetylating the chromatin. The catalytic activity of HDACs is mainly dependent on its incorporation into multiprotein co-repressor complexes, among which SIN3A-HDAC2 co-repressor is widely studied to regulate synaptic plasticity. However, the involvement of co-repressor complex in regulating memory loss or amnesia is unexplored. This study examines the role of co-repressor SIN3A in scopolamine-induced amnesia through epigenetic changes in the hippocampus. Scopolamine treatment remarkably enhanced hippocampal SIN3A expression in mice. To prevent such increase in SIN3A expression, we used hippocampal infusion of SIN3A-siRNA and assessed the effect of SIN3A silencing on scopolamine-induced amnesia. Silencing of SIN3A in amnesic mice reduced the binding of HDAC2 at neuronal immediate early genes (IEGs) promoter, but did not change the expression of HDAC2. Furthermore, it increased acetylation of H3K9 and H3K14 at neuronal IEGs (Arc, Egr1, Homer1 and Narp) promoter, prevented scopolamine-induced down-regulation of IEGs and improved consolidation of memory during novel object recognition task. These findings together suggest that SIN3A has a critical role in regulation of synaptic plasticity and might act as a potential therapeutic target to rescue memory decline during amnesia and other neuropsychiatric pathologies. © 2018 International Society for Neurochemistry.

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

PubMed

Corthésy, B; Cardinaux, J R; Claret, F X; Wahli, W

1989-12-01

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.

Identification and characterization of PhbF: a DNA binding protein with regulatory role in the PHB metabolism of Herbaspirillum seropedicae SmR1.

PubMed

Kadowaki, Marco A S; Müller-Santos, Marcelo; Rego, Fabiane G M; Souza, Emanuel M; Yates, Marshall G; Monteiro, Rose A; Pedrosa, Fabio O; Chubatsu, Leda S; Steffens, Maria B R

2011-10-14

Herbaspirillum seropedicae SmR1 is a nitrogen fixing endophyte associated with important agricultural crops. It produces polyhydroxybutyrate (PHB) which is stored intracellularly as granules. However, PHB metabolism and regulatory control is not yet well studied in this organism. In this work we describe the characterization of the PhbF protein from H. seropedicae SmR1 which was purified and characterized after expression in E. coli. The purified PhbF protein was able to bind to eleven putative promoters of genes involved in PHB metabolism in H. seropedicae SmR1. In silico analyses indicated a probable DNA-binding sequence which was shown to be protected in DNA footprinting assays using purified PhbF. Analyses using lacZ fusions showed that PhbF can act as a repressor protein controlling the expression of PHB metabolism-related genes. Our results indicate that H. seropedicae SmR1 PhbF regulates expression of phb-related genes by acting as a transcriptional repressor. The knowledge of the PHB metabolism of this plant-associated bacterium may contribute to the understanding of the plant-colonizing process and the organism's resistance and survival in planta.

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes

PubMed Central

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-01-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression. PMID:22434878

Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes.

PubMed

Metz, Sebastian; Haberzettl, Kerstin; Frühwirth, Sebastian; Teich, Kristin; Hasewinkel, Christian; Klug, Gabriele

2012-07-01

The expression of photosynthesis genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by the oxygen tension and by light quantity. Two photoreceptor proteins, AppA and CryB, have been identified in the past, which are involved in this regulation. AppA senses light by its N-terminal BLUF domain, its C-terminal part binds heme and is redox-responsive. Through its interaction to the transcriptional repressor PpsR the AppA photoreceptor controls expression of photosynthesis genes. The cryptochrome-like protein CryB was shown to affect regulation of photosynthesis genes, but the underlying signal chain remained unknown. Here we show that CryB interacts with the C-terminal domain of AppA and modulates the binding of AppA to the transcriptional repressor PpsR in a light-dependent manner. Consequently, binding of the transcription factor PpsR to its DNA target is affected by CryB. In agreement with this, all genes of the PpsR regulon showed altered expression levels in a CryB deletion strain after blue-light illumination. These results elucidate for the first time how a bacterial cryptochrome affects gene expression.

ThrR, a DNA-binding transcription factor involved in controlling threonine biosynthesis in Bacillus subtilis.

PubMed

Rosenberg, Jonathan; Müller, Peter; Lentes, Sabine; Thiele, Martin J; Zeigler, Daniel R; Tödter, Dominik; Paulus, Henry; Brantl, Sabine; Stülke, Jörg; Commichau, Fabian M

2016-09-01

The threonine dehydratase IlvA is part of the isoleucine biosynthesis pathway in the Gram-positive model bacterium Bacillus subtilis. Consequently, deletion of ilvA causes isoleucine auxotrophy. It has been reported that ilvA pseudo-revertants having a derepressed hom-thrCB operon appear in the presence of threonine. Here we have characterized two classes of ilvA pseudo-revertants. In the first class the hom-thrCB operon was derepressed unmasking the threonine dehydratase activity of the threonine synthase ThrC. In the second class of mutants, threonine biosynthesis was more broadly affected. The first class of ilvA pseudo-revertants had a mutation in the Phom promoter (P*hom ), resulting in constitutive expression of the hom-thrCB operon. In the second class of ilvA pseudo-revertants, the thrR gene encoding a putative DNA-binding protein was inactivated, also resulting in constitutive expression of the hom-thrCB operon. Here we demonstrate that ThrR is indeed a DNA-binding transcription factor that regulates the hom-thrCB operon and the thrD aspartokinase gene. DNA binding assays uncovered the DNA-binding site of ThrR and revealed that the repressor competes with the RNA polymerase for DNA binding. This study also revealed that ThrR orthologs are ubiquitous in genomes from the Gram-positive phylum Firmicutes and in some Gram-negative bacteria. © 2016 John Wiley & Sons Ltd.

The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle.

PubMed

Pramila, Tata; Wu, Wei; Miles, Shawna; Noble, William Stafford; Breeden, Linda L

2006-08-15

Transcription patterns shift dramatically as cells transit from one phase of the cell cycle to another. To better define this transcriptional circuitry, we collected new microarray data across the cell cycle of budding yeast. The combined analysis of these data with three other cell cycle data sets identifies hundreds of new highly periodic transcripts and provides a weighted average peak time for each transcript. Using these data and phylogenetic comparisons of promoter sequences, we have identified a late S-phase-specific promoter element. This element is the binding site for the forkhead protein Hcm1, which is required for its cell cycle-specific activity. Among the cell cycle-regulated genes that contain conserved Hcm1-binding sites, there is a significant enrichment of genes involved in chromosome segregation, spindle dynamics, and budding. This may explain why Hcm1 mutants show 10-fold elevated rates of chromosome loss and require the spindle checkpoint for viability. Hcm1 also induces the M-phase-specific transcription factors FKH1, FKH2, and NDD1, and two cell cycle-specific transcriptional repressors, WHI5 and YHP1. As such, Hcm1 fills a significant gap in our understanding of the transcriptional circuitry that underlies the cell cycle.

SOX2 as a New Regulator of HPV16 Transcription.

PubMed

Martínez-Ramírez, Imelda; Del-Castillo-Falconi, Víctor; Mitre-Aguilar, Irma B; Amador-Molina, Alfredo; Carrillo-García, Adela; Langley, Elizabeth; Zentella-Dehesa, Alejandro; Soto-Reyes, Ernesto; García-Carrancá, Alejandro; Herrera, Luis A; Lizano, Marcela

2017-07-05

Persistent infections with high-risk human papillomavirus (HPV) constitute the main risk factor for cervical cancer development. HPV16 is the most frequent type associated to squamous cell carcinomas (SCC), followed by HPV18. The long control region (LCR) in the HPV genome contains the replication origin and sequences recognized by cellular transcription factors (TFs) controlling viral transcription. Altered expression of E6 and E7 viral oncogenes, modulated by the LCR, causes modifications in cellular pathways such as proliferation, leading to malignant transformation. The aim of this study was to identify specific TFs that could contribute to the modulation of high-risk HPV transcriptional activity, related to the cellular histological origin. We identified sex determining region Y (SRY)-box 2 (SOX2) response elements present in HPV16-LCR. SOX2 binding to the LCR was demonstrated by in vivo and in vitro assays. The overexpression of this TF repressed HPV16-LCR transcriptional activity, as shown through reporter plasmid assays and by the down-regulation of endogenous HPV oncogenes. Site-directed mutagenesis revealed that three putative SOX2 binding sites are involved in the repression of the LCR activity. We propose that SOX2 acts as a transcriptional repressor of HPV16-LCR, decreasing the expression of E6 and E7 oncogenes in a SCC context.

Pirating conserved phage mechanisms promotes promiscuous staphylococcal pathogenicity island transfer.

PubMed

Bowring, Janine; Neamah, Maan M; Donderis, Jorge; Mir-Sanchis, Ignacio; Alite, Christian; Ciges-Tomas, J Rafael; Maiques, Elisa; Medmedov, Iltyar; Marina, Alberto; Penadés, José R

2017-08-08

Targeting conserved and essential processes is a successful strategy to combat enemies. Remarkably, the clinically important Staphylococcus aureus pathogenicity islands (SaPIs) use this tactic to spread in nature. SaPIs reside passively in the host chromosome, under the control of the SaPI-encoded master repressor, Stl. It has been assumed that SaPI de-repression is effected by specific phage proteins that bind to Stl, initiating the SaPI cycle. Different SaPIs encode different Stl repressors, so each targets a specific phage protein for its de-repression. Broadening this narrow vision, we report here that SaPIs ensure their promiscuous transfer by targeting conserved phage mechanisms. This is accomplished because the SaPI Stl repressors have acquired different domains to interact with unrelated proteins, encoded by different phages, but in all cases performing the same conserved function. This elegant strategy allows intra- and inter-generic SaPI transfer, highlighting these elements as one of nature's most fascinating subcellular parasites.

Strong linkage disequilibrium of a HbE variant with the (AT)9(T)5 repeat in the BP1 binding site upstream of the beta-globin gene in the Thai population.

PubMed

Ohashi, Jun; Naka, Izumi; Patarapotikul, Jintana; Hananantachai, Hathairad; Brittenham, Gary; Looareesuwan, Sornchai; Clark, Andrew G; Tokunaga, Katsushi

2005-01-01

A binding site for the repressor protein BP1, which contains a tandem (AT)x(T)y repeat, is located approximately 530 bp 5' to the human beta-globin gene (HBB). There is accumulating evidence that BP1 binds to the (AT)9(T)5 allele more strongly than to other alleles, thereby reducing the expression of HBB. In this study, we investigated polymorphisms in the (AT)x(T)y repeat in 57 individuals living in Thailand, including three homozygotes for the hemoglobin E variant (HbE; beta26Glu-->Lys), 22 heterozygotes, and 32 normal homozygotes. We found that (AT)9(T)5 and (AT)7(T)7 alleles were predominant in the studied population and that the HbE variant is in strong linkage disequilibrium with the (AT)9(T)5 allele, which can explain why the betaE chain is inefficiently synthesized compared to the normal betaA chain. Moreover, the mildness of the HbE disease compared to other hemoglobinopathies in Thai may be due, in part, to the presence of the (AT)9(T)5 repeat on the HbE chromosome. In addition, a novel (AC)n polymorphism adjacent to the (AT)x(T)y repeat (i.e., (AC)3(AT)7(T)5) was found through the variation screening in this study.

GENE REGULATION. Discrete functions of nuclear receptor Rev-erbα couple metabolism to the clock.

PubMed

Zhang, Yuxiang; Fang, Bin; Emmett, Matthew J; Damle, Manashree; Sun, Zheng; Feng, Dan; Armour, Sean M; Remsberg, Jarrett R; Jager, Jennifer; Soccio, Raymond E; Steger, David J; Lazar, Mitchell A

2015-06-26

Circadian and metabolic physiology are intricately intertwined, as illustrated by Rev-erbα, a transcription factor (TF) that functions both as a core repressive component of the cell-autonomous clock and as a regulator of metabolic genes. Here, we show that Rev-erbα modulates the clock and metabolism by different genomic mechanisms. Clock control requires Rev-erbα to bind directly to the genome at its cognate sites, where it competes with activating ROR TFs. By contrast, Rev-erbα regulates metabolic genes primarily by recruiting the HDAC3 co-repressor to sites to which it is tethered by cell type-specific transcription factors. Thus, direct competition between Rev-erbα and ROR TFs provides a universal mechanism for self-sustained control of the molecular clock across all tissues, whereas Rev-erbα uses lineage-determining factors to convey a tissue-specific epigenomic rhythm that regulates metabolism tailored to the specific need of that tissue. Copyright © 2015, American Association for the Advancement of Science.

Structural Basis for NADH/NAD+ Redox Sensing by a Rex Family Repressor

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

McLaughlin, K.J.; Soares, A.; Strain-Damerell, C. M.

2010-05-28

Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD{sup +} is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD{sup +} ratio. Here we investigate the molecular mechanism for NADH/NAD{sup +} sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD{sup +}, (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rexmore » from the DNA site following a 40{sup o} closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.« less

The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis*

PubMed Central

Ho, Ngoc Anh Thu; Dawes, Stephanie S.; Crowe, Adam M.; Casabon, Israël; Gao, Chen; Kendall, Sharon L.; Baker, Edward N.; Eltis, Lindsay D.; Lott, J. Shaun

2016-01-01

Cholesterol can be a major carbon source for Mycobacterium tuberculosis during infection, both at an early stage in the macrophage phagosome and later within the necrotic granuloma. KstR is a highly conserved TetR family transcriptional repressor that regulates a large set of genes responsible for cholesterol catabolism. Many genes in this regulon, including kstR, are either induced during infection or are essential for survival of M. tuberculosis in vivo. In this study, we identified two ligands for KstR, both of which are CoA thioester cholesterol metabolites with four intact steroid rings. A metabolite in which one of the rings was cleaved was not a ligand. We confirmed the ligand-protein interactions using intrinsic tryptophan fluorescence and showed that ligand binding strongly inhibited KstR-DNA binding using surface plasmon resonance (IC50 for ligand = 25 nm). Crystal structures of the ligand-free form of KstR show variability in the position of the DNA-binding domain. In contrast, structures of KstR·ligand complexes are highly similar to each other and demonstrate a position of the DNA-binding domain that is unfavorable for DNA binding. Comparison of ligand-bound and ligand-free structures identifies residues involved in ligand specificity and reveals a distinctive mechanism by which the ligand-induced conformational change mediates DNA release. PMID:26858250

Modulation of DNA binding by gene-specific transcription factors.

PubMed

Schleif, Robert F

2013-10-01

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

Transcriptional activation of NAD+-dependent protein deacetylase SIRT1 by nuclear receptor TLX.

PubMed

Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

2009-09-04

An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD(+)-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX

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

Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi

2009-09-04

An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis

PubMed Central

Henriksson-Peltola, Petri; Sehlén, Wilhelmina; Haggård-Ljungquist, Elisabeth

2007-01-01

Bacteriophages P2, P2 Hy dis and WΦ are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein–DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein–DNA complexes, and only WΦ was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WΦ operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and WΦ C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages. PMID:17412705

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.

Metal Binding Studies and EPR Spectroscopy of the Manganese Transport Regulator MntR†

PubMed Central

Golynskiy, Misha V.; Gunderson, William A.; Hendrich, Michael P.; Cohen, Seth M.

2007-01-01

Manganese transport regulator (MntR) is a member of the diphtheria toxin repressor (DtxR) family of transcription factors that is responsible for manganese homeostasis in Bacillus subtilis. Prior biophysical studies have focused on the metal-mediated DNA binding of MntR [Lieser, S. A., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2003) Biochemistry 42, 12634-12642], as well as metal stabilization of the MntR structure [Golynskiy, M. V., Davis, T. C., Helmann, J. D., and Cohen, S. M. (2005) Biochemistry 44, 3380-3389], but only limited data on the metal-binding affinities for MntR are available. Herein, the metal-binding affinities of MntR were determined by using electron paramagnetic resonance (EPR) spectroscopy, as well as competition experiments with the fluorimetric dyes Fura-2 and Mag-fura-2. MntR was not capable of competing with Fura-2 for the binding of transition metal ions. Therefore, the metal-binding affinities and stoichiometries of Mag-fura-2 for Mn2+, Co2+, Ni2+, Zn2+, and Cd2+ were determined and utilized in MntR/Mag-fura-2 competition experiments. The measured Kd values for MntR metal binding are comparable to those reported for DtxR metal binding [Kd from 10-7 to 10-4 M; D’Aquino, J. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 18408-18413], AntR [a homologue from Bacillus anthracis; Sen, K. I. et al. (2006) Biochemistry 45, 4295-4303], and generally follow the Irving-Williams series. Direct detection of the dinuclear Mn2+ site in MntR with EPR spectroscopy is presented, and the exchange interaction was determined, J = -0.2 cm-1. This value is lower in magnitude than most known dinuclear Mn2+ sites in proteins and synthetic complexes and is consistent with a dinuclear Mn2+ site with a longer Mn···Mn distance (4.4 Å) observed in some of the available crystal structures. MntR is found to have a surprisingly low binding affinity (∼160 μM) for its cognate metal ion Mn2+. Moreover, the results of DNA binding studies in the presence of limiting metal ion concentrations were found to be consistent with the measured metal-binding constants. The metal-binding affinities of MntR reported here help to elucidate the regulatory mechanism of this metal-dependent transcription factor. PMID:17176058

Structural Basis of Transcriptional Regulation of the Proline Utilization Regulon by Multifunctional PutA

PubMed Central

Zhou, Yuzhen; Larson, John D.; Bottoms, Christopher A.; Arturo, Emilia C.; Henzl, Michael T.; Jenkins, Jermaine L.; Nix, Jay C.; Becker, Donald F.; Tanner, John J.

2009-01-01

Summary The multifunctional Escherichia coli PutA flavoprotein functions as both a membrane-associated proline catabolic enzyme and transcriptional repressor of the proline utilization genes putA and putP. To better understand the mechanism of transcriptional regulation by PutA, we have mapped the put regulatory region, determined a crystal structure of the PutA ribbon-helix-helix domain (PutA52) complexed with DNA and examined the thermodynamics of DNA binding to PutA52. Five operator sites, each containing the sequence motif 5′-GTTGCA-3′, were identified using gel-shift analysis. Three of the sites are shown to be critical for repression of putA, whereas the two other sites are important for repression of putP. The 2.25 Å resolution crystal structure of PutA52 bound to one of the operators (operator 2, 21-bp) shows that the protein contacts a 9-bp fragment, corresponding to the GTTGCA consensus motif plus three flanking base pairs. Since the operator sequences differ in flanking bases, the structure implies that PutA may have different affinities for the five operators. This hypothesis was explored using isothermal titration calorimetry. The binding of PutA52 to operator 2 is exothermic with an enthalpy of −1.8 kcal/mol and a dissociation constant of 210 nM. Substitution of the flanking bases of operator 4 into operator 2 results in an unfavorable enthalpy of 0.2 kcal/mol and 15-fold lower affinity, which shows that base pairs outside of the consensus motif impact binding. The structural and thermodynamic data suggest that hydrogen bonds between Lys9 and bases adjacent to the GTTGCA motif contribute to transcriptional regulation by fine-tuning the affinity of PutA for put control operators. PMID:18586269

Solution NMR Refinement of a Metal Ion Bound Protein Using Metal Ion Inclusive Restrained Molecular Dynamics Methods

PubMed Central

Chakravorty, Dhruva K.; Wang, Bing; Lee, Chul Won; Guerra, Alfredo J.; Giedroc, David P.; Merz, Kenneth M.

2013-01-01

Correctly calculating the structure of metal coordination sites in a protein during the process of nuclear magnetic resonance (NMR) structure determination and refinement continues to be a challenging task. In this study, we present an accurate and convenient means by which to include metal ions in the NMR structure determination process using molecular dynamics (MD) constrained by NMR-derived data to obtain a realistic and physically viable description of the metal binding site(s). This method provides the framework to accurately portray the metal ions and its binding residues in a pseudo-bond or dummy-cation like approach, and is validated by quantum mechanical/molecular mechanical (QM/MM) MD calculations constrained by NMR-derived data. To illustrate this approach, we refine the zinc coordination complex structure of the zinc sensing transcriptional repressor protein Staphylococcus aureus CzrA, generating over 130 ns of MD and QM/MM MD NMR-data compliant sampling. In addition to refining the first coordination shell structure of the Zn(II) ion, this protocol benefits from being performed in a periodically replicated solvation environment including long-range electrostatics. We determine that unrestrained (not based on NMR data) MD simulations correlated to the NMR data in a time-averaged ensemble. The accurate solution structure ensemble of the metal-bound protein accurately describes the role of conformational dynamics in allosteric regulation of DNA binding by zinc and serves to validate our previous unrestrained MD simulations of CzrA. This methodology has potentially broad applicability in the structure determination of metal ion bound proteins, protein folding and metal template protein-design studies. PMID:23609042

BEND3 is involved in the human-specific repression of calreticulin: Implication for the evolution of higher brain functions in human.

PubMed

Aghajanirefah, A; Nguyen, L N; Ohadi, M

2016-01-15

Recent emerging evidence indicates that changes in gene expression levels are linked to human evolution. We have previously reported a human-specific nucleotide in the promoter sequence of the calreticulin (CALR) gene at position -220C, which is the site of action of valproic acid. Reversion of this nucleotide to the ancestral A-allele has been detected in patients with degrees of deficit in higher brain cognitive functions. This mutation has since been reported in the 1000 genomes database at an approximate frequency of <0.0004 in humans (rs138452745). In the study reported here, we present update on the status of rs138452745 across evolution, based on the Ensembl and NCBI databases. The DNA pulldown assay was also used to identify the proteins binding to the C- and A-alleles, using two cell lines, SK-N-BE and HeLa. Consistent with our previous findings, the C-allele is human-specific, and the A-allele is the rule across all other species (N=38). This nucleotide resides in a block of 12-nucleotides that is strictly conserved across evolution. The DNA pulldown experiments revealed that in both SK-N-BE and HeLa cells, the transcription repressor BEN domain containing 3 (BEND3) binds to the human-specific C-allele, whereas the nuclear factor I (NFI) family members, NF1A, B, C, and X, specifically bind to the ancestral A-allele. This binding pattern is consistent with a previously reported decreased promoter activity of the C-allele vs. the A-allele. We propose that there is a link between binding of BEND3 to the CALR rs138452745 C-allele and removal of NFI binding site from this nucleotide, and the evolution of human-specific higher brain functions. To our knowledge, CALR rs138452745 is the first instance of enormous nucleotide conservation across evolution, except in the human species. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification of a Signal-Responsive Nuclear Export Sequence in Class II Histone Deacetylases

PubMed Central

McKinsey, Timothy A.; Zhang, Chun Li; Olson, Eric N.

2001-01-01

Activation of muscle-specific genes by the MEF2 transcription factor is inhibited by class II histone deacetylases (HDACs) 4 and 5, which contain carboxy-terminal deacetylase domains and amino-terminal extensions required for association with MEF2. The inhibitory action of HDACs is overcome by myogenic signals which disrupt MEF2-HDAC interactions and stimulate nuclear export of these transcriptional repressors. Nucleocytoplasmic trafficking of HDAC5 is mediated by binding of the chaperone protein 14-3-3 to two phosphoserine residues (Ser-259 and Ser-498) in its amino-terminal extension. Here we show that HDAC4 and -5 each contain a signal-responsive nuclear export sequence (NES) at their extreme carboxy termini. The NES is conserved in another class II HDAC, HDAC7, but is absent in class I HDACs and the HDAC-related corepressor, MEF2-interacting transcription repressor. Our results suggest that this conserved NES is inactive in unphosphorylated HDAC5, which is localized to the nucleus, and that calcium-calmodulin-dependent protein kinase (CaMK)-dependent binding of 14-3-3 to phosphoserines 259 and 498 activates the NES, with consequent export of the transcriptional repressor to the cytoplasm. A single amino acid substitution in this NES is sufficient to retain HDAC5 in the nucleus in the face of CaMK signaling. These findings provide molecular insight into the mechanism by which extracellular cues alter chromatin structure to promote muscle differentiation and other MEF2-regulated processes. PMID:11509672

LWD-TCP complex activates the morning gene CCA1 in Arabidopsis.

PubMed

Wu, Jing-Fen; Tsai, Huang-Lung; Joanito, Ignasius; Wu, Yi-Chen; Chang, Chin-Wen; Li, Yi-Hang; Wang, Ying; Hong, Jong Chan; Chu, Jhih-Wei; Hsu, Chao-Ping; Wu, Shu-Hsing

2016-10-13

A double-negative feedback loop formed by the morning genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) and the evening gene TIMING OF CAB EXPRESSION1 (TOC1) contributes to regulation of the circadian clock in Arabidopsis. A 24-h circadian cycle starts with the peak expression of CCA1 at dawn. Although CCA1 is targeted by multiple transcriptional repressors, including PSEUDO-RESPONSE REGULATOR9 (PRR9), PRR7, PRR5 and CCA1 HIKING EXPEDITION (CHE), activators of CCA1 remain elusive. Here we use mathematical modelling to infer a co-activator role for LIGHT-REGULATED WD1 (LWD1) in CCA1 expression. We show that the TEOSINTE BRANCHED 1-CYCLOIDEA-PCF20 (TCP20) and TCP22 proteins act as LWD-interacting transcriptional activators. The concomitant binding of LWD1 and TCP20/TCP22 to the TCP-binding site in the CCA1 promoter activates CCA1. Our study reveals activators of the morning gene CCA1 and provides an action mechanism that ensures elevated expression of CCA1 at dawn to sustain a robust clock.

LWD–TCP complex activates the morning gene CCA1 in Arabidopsis

PubMed Central

Wu, Jing-Fen; Tsai, Huang-Lung; Joanito, Ignasius; Wu, Yi-Chen; Chang, Chin-Wen; Li, Yi-Hang; Wang, Ying; Hong, Jong Chan; Chu, Jhih-Wei; Hsu, Chao-Ping; Wu, Shu-Hsing

2016-01-01

A double-negative feedback loop formed by the morning genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) and the evening gene TIMING OF CAB EXPRESSION1 (TOC1) contributes to regulation of the circadian clock in Arabidopsis. A 24-h circadian cycle starts with the peak expression of CCA1 at dawn. Although CCA1 is targeted by multiple transcriptional repressors, including PSEUDO-RESPONSE REGULATOR9 (PRR9), PRR7, PRR5 and CCA1 HIKING EXPEDITION (CHE), activators of CCA1 remain elusive. Here we use mathematical modelling to infer a co-activator role for LIGHT-REGULATED WD1 (LWD1) in CCA1 expression. We show that the TEOSINTE BRANCHED 1-CYCLOIDEA-PCF20 (TCP20) and TCP22 proteins act as LWD-interacting transcriptional activators. The concomitant binding of LWD1 and TCP20/TCP22 to the TCP-binding site in the CCA1 promoter activates CCA1. Our study reveals activators of the morning gene CCA1 and provides an action mechanism that ensures elevated expression of CCA1 at dawn to sustain a robust clock. PMID:27734958

Development of a formaldehyde biosensor with application to synthetic methylotrophy.

PubMed

Woolston, Benjamin M; Roth, Timothy; Kohale, Ishwar; Liu, David R; Stephanopoulos, Gregory

2018-01-01

Formaldehyde is a prevalent environmental toxin and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is, therefore, of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of Escherichia coli. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 µM. We then used the sensor to benchmark the in vivo activity of several NAD-dependent methanol dehydrogenase (Mdh) variants, the rate-limiting enzyme that catalyzes the first step of methanol assimilation. In order to use this biosensor to distinguish individuals in a mixed population of Mdh variants, we developed a strategy to prevent cross-talk by using glutathione as a formaldehyde sink to minimize intercellular formaldehyde diffusion. Finally, we applied this biosensor to balance expression of mdh and the formaldehyde assimilation enzymes hps and phi in an engineered E. coli strain to minimize formaldehyde build-up while also reducing the burden of heterologous expression. This biosensor offers a quick and simple method for sensitively detecting formaldehyde, and has the potential to be used as the basis for directed evolution of Mdh and dynamic formaldehyde control strategies for establishing synthetic methylotrophy. © 2017 Wiley Periodicals, Inc.

Regulation of the sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC 824 by a Putative Transcriptional Repressor

PubMed Central

Nair, Ramesh V.; Green, Edward M.; Watson, David E.; Bennett, George N.; Papoutsakis, Eleftherios T.

1999-01-01

A gene (orf1, now designated solR) previously identified upstream of the aldehyde/alcohol dehydrogenase gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 176:871–885, 1994) was found to encode a repressor of the sol locus (aad, ctfA, ctfB and adc) genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824. Primer extension analysis identified a transcriptional start site 35 bp upstream of the solR start codon. Amino acid comparisons of SolR identified a potential helix-turn-helix DNA-binding motif in the C-terminal half towards the center of the protein, suggesting a regulatory role. Overexpression of SolR in strain ATCC 824(pCO1) resulted in a solvent-negative phenotype owing to its deleterious effect on the transcription of the sol locus genes. Inactivation of solR in C. acetobutylicum via homologous recombination yielded mutants B and H (ATCC 824 solR::pO1X) which exhibited deregulated solvent production characterized by increased flux towards butanol and acetone formation, earlier induction of aad, lower overall acid production, markedly improved yields of solvents on glucose, a prolonged solvent production phase, and increased biomass accumulation compared to those of the wild-type strain. PMID:9864345

A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit

PubMed Central

2017-01-01

Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range. PMID:29084263

Control of alternative splicing by forskolin through hnRNP K during neuronal differentiation

PubMed Central

Cao, Wenguang; Razanau, Aleh; Feng, Dairong; Lobo, Vincent G.; Xie, Jiuyong

2012-01-01

The molecular basis of cell signal-regulated alternative splicing at the 3′ splice site remains largely unknown. We isolated a protein kinase A-responsive ribonucleic acid (RNA) element from a 3′ splice site of the synaptosomal-associated protein 25 (Snap25) gene for forskolin-inhibited splicing during neuronal differentiation of rat pheochromocytoma PC12 cells. The element binds specifically to heterogeneous nuclear ribonucleo protein (hnRNP) K in a phosphatase-sensitive way, which directly competes with the U2 auxiliary factor U2AF65, an essential component of early spliceosomes. Transcripts with similarly localized hnRNP K target motifs upstream of alternative exons are enriched in genes often associated with neurological diseases. We show that such motifs upstream of the Runx1 exon 6 also bind hnRNP K, and importantly, hnRNP K is required for forskolin-induced repression of the exon. Interestingly, this exon encodes the peptide domain that determines the switch of the transcriptional repressor/activator activity of Runx1, a change known to be critical in specifying neuron lineages. Consistent with an important role of the target genes in neurons, knocking down hnRNP K severely disrupts forskolin-induced neurite growth. Thus, through hnRNP K, the neuronal differentiation stimulus forskolin targets a critical 3′ splice site component of the splicing machinery to control alternative splicing of crucial genes. This also provides a regulated direct competitor of U2AF65 for cell signal control of 3′ splice site usage. PMID:22684629

Nuclear IL-33 regulates soluble ST2 receptor and IL-6 expression in primary human arterial endothelial cells and is decreased in idiopathic pulmonary arterial hypertension

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

Shao, Dongmin; Perros, Frédéric; Caramori, Gaetano

Highlights: • Nuclear IL-33 expression is reduced in vascular endothelial cells from PAH patients. • Knockdown of IL-33 leads to increased IL-6 and sST2 mRNA expression. • IL-33 binds homeobox motifs in target gene promoters and recruits repressor proteins. - Abstract: Idiopathic pulmonary arterial hypertension (IPAH) is an incurable condition leading to right ventricular failure and death and inflammation is postulated to be associated with vascular remodelling. Interleukin (IL)-33, a member of the “alarmin” family can either act on the membrane ST2 receptor or as a nuclear repressor, to regulate inflammation. We show, using immunohistochemistry, that IL-33 expression is nuclearmore » in the vessels of healthy subjects whereas nuclear IL-33 is markedly diminished in the vessels of IPAH patients. This correlates with reduced IL-33 mRNA expression in their lung. In contrast, serum levels of IL-33 are unchanged in IPAH. However, the expression of the soluble form of ST2, sST2, is enhanced in the serum of IPAH patients. Knock-down of IL-33 in human endothelial cells (ECs) using siRNA is associated with selective modulation of inflammatory genes involved in vascular remodelling including IL-6. Additionally, IL-33 knock-down significantly increased sST2 release from ECs. Chromatin immunoprecipitation demonstrated that IL-33 bound multiple putative homeodomain protein binding motifs in the proximal and distal promoters of ST2 genes. IL-33 formed a complex with the histone methyltransferase SUV39H1, a transcriptional repressor. In conclusion, IL-33 regulates the expression of IL-6 and sST2, an endogenous IL-33 inhibitor, in primary human ECs and may play an important role in the pathogenesis of PAH through recruitment of transcriptional repressor proteins.« less

ANKRD1 Acts as a Transcriptional Repressor of MMP13 via the AP-1 Site

PubMed Central

Almodóvar-García, Karinna; Kwon, Minjae; Samaras, Susan E.

2014-01-01

The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1−/− (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1fl/fl (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs. PMID:24515436

Evolution of context dependent regulation by expansion of feast/famine regulatory proteins

DOE PAGES

Plaisier, Christopher L.; Lo, Fang -Yin; Ashworth, Justin; ...

2014-11-14

Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions thatmore » mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.« less

Evolution of context dependent regulation by expansion of feast/famine regulatory proteins.

PubMed

Plaisier, Christopher L; Lo, Fang-Yin; Ashworth, Justin; Brooks, Aaron N; Beer, Karlyn D; Kaur, Amardeep; Pan, Min; Reiss, David J; Facciotti, Marc T; Baliga, Nitin S

2014-11-14

Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions that mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.

The translational repressor Crc controls the Pseudomonas putida benzoate and alkane catabolic pathways using a multi-tier regulation strategy.

PubMed

Hernández-Arranz, Sofía; Moreno, Renata; Rojo, Fernando

2013-01-01

Metabolically versatile bacteria usually perceive aromatic compounds and hydrocarbons as non-preferred carbon sources, and their assimilation is inhibited if more preferable substrates are available. This is achieved via catabolite repression. In Pseudomonas putida, the expression of the genes allowing the assimilation of benzoate and n-alkanes is strongly inhibited by catabolite repression, a process controlled by the translational repressor Crc. Crc binds to and inhibits the translation of benR and alkS mRNAs, which encode the transcriptional activators that induce the expression of the benzoate and alkane degradation genes respectively. However, sequences similar to those recognized by Crc in benR and alkS mRNAs exist as well in the translation initiation regions of the mRNA of several structural genes of the benzoate and alkane pathways, which suggests that Crc may also regulate their translation. The present results show that some of these sites are functional, and that Crc inhibits the induction of both pathways by limiting not only the translation of their transcriptional activators, but also that of genes coding for the first enzyme in each pathway. Crc may also inhibit the translation of a gene involved in benzoate uptake. This multi-tier approach probably ensures the rapid regulation of pathway genes, minimizing the assimilation of non-preferred substrates when better options are available. A survey of possible Crc sites in the mRNAs of genes associated with other catabolic pathways suggested that targeting substrate uptake, pathway induction and/or pathway enzymes may be a common strategy to control the assimilation of non-preferred compounds. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Human T-cell leukemia virus type I oncoprotein Tax represses Smad-dependent transforming growth factor beta signaling through interaction with CREB-binding protein/p300.

PubMed

Mori, N; Morishita, M; Tsukazaki, T; Giam, C Z; Kumatori, A; Tanaka, Y; Yamamoto, N

2001-04-01

Human T-cell leukemia virus type I (HTLV-I) Tax is a potent transcriptional regulator that can activate or repress specific cellular genes and that has been proposed to contribute to leukemogenesis in adult T-cell leukemia. Previously, HTLV-I- infected T-cell clones were found to be resistant to growth inhibition by transforming growth factor (TGF)-beta. Here it is shown that Tax can perturb Smad-dependent TGF-beta signaling even though no direct interaction of Tax and Smad proteins could be detected. Importantly, a mutant Tax of CREB-binding protein (CBP)/p300 binding site, could not repress the Smad transactivation function, suggesting that the CBP/p300 binding domain of Tax is essential for the suppression of Smad function. Because both Tax and Smad are known to interact with CBP/p300 for the potentiation of their transcriptional activities, the effect of CBP/p300 on suppression of Smad-mediated transactivation by Tax was examined. Overexpression of CBP/p300 reversed Tax-mediated inhibition of Smad transactivation. Furthermore, Smad could repress Tax transcriptional activation, indicating reciprocal repression between Tax and Smad. These results suggest that Tax interferes with the recruitment of CBP/p300 into transcription initiation complexes on TGF-beta-responsive elements through its binding to CBP/p300. The novel function of Tax as a repressor of TGF-beta signaling may contribute to HTLV-I leukemogenesis. (Blood. 2001;97:2137-2144)

The transcriptional repressor DREAM is involved in thyroid gene expression

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

D'Andrea, Barbara; Di Palma, Tina; Mascia, Anna

2005-04-15

Downstream regulatory element antagonistic modulator (DREAM) was originally identified in neuroendocrine cells as a calcium-binding protein that specifically binds to downstream regulatory elements (DRE) on DNA, and represses transcription of its target genes. To explore the possibility that DREAM may regulate the endocrine activity of the thyroid gland, we analyzed its mRNA expression in undifferentiated and differentiated thyroid cells. We demonstrated that DREAM is expressed in the normal thyroid tissue as well as in differentiated thyroid cells in culture while it is absent in FRT poorly differentiated cells. In the present work, we also show that DREAM specifically binds tomore » DRE sites identified in the 5' untranslated region (UTR) of the thyroid-specific transcription factors Pax8 and TTF-2/FoxE1 in a calcium-dependent manner. By gel retardation assays we demonstrated that thapsigargin treatment increases the binding of DREAM to the DRE sequences present in Pax8 and TTF-2/Foxe1 5' UTRs, and this correlates with a significant reduction of the expression of these genes. Interestingly, in poorly differentiated thyroid cells overexpression of exogenous DREAM strongly inhibits Pax8 expression. Moreover, we provide evidence that a mutated form of DREAM unable to bind Ca{sup 2+} interferes with thyroid cell proliferation. Therefore, we propose that in thyroid cells DREAM is a mediator of the calcium-signaling pathway and it is involved in the regulation of thyroid cell function.« less

Allosteric transcriptional regulation via changes in the overall topology of the core promoter

DOE PAGES

Philips, Steven J.; Canalizo-Hernandez, Monica; Yildirim, Ilyas; ...

2015-08-21

Many transcriptional activators act at a distance from core promoter elements and work by recruiting RNA polymerase through protein-protein interactions. We show here how the prokaryotic regulatory protein CueR both represses and activates transcription by differentially modulating local DNA structure within the promoter. Structural studies reveal that the repressor state slightly bends the promoter DNA, precluding optimal RNA polymerase-promoter recognition. Upon binding a metal ion in the allosteric site, CueR switches into an activator conformation. It maintains all protein-DNA contacts but introduces torsional stresses that kink and undertwist the promoter, stabilizing an A-DNA-like conformation. Finally, these factors switch on andmore » off transcription by exerting dynamic control of DNA stereochemistry, reshaping the core promoter and making it a better or worse substrate for polymerase.« less

Purification and characterization of human pancreatic polypeptide expressed in E. coli.

PubMed

Griko, Y V; Kapanadze, M D

1995-08-04

The region of cDNA encoding human pancreatic polypeptide (hPP) was obtained by polymerase chain reaction (PCR) and subcloned into an expression vector. The pancreatic polypeptide gene was expressed in Escherichia coli in two versions: as a cleavable fusion protein with IgG-binding synthetic ZZ domains of protein A from Staphylococcus aureus or with the 1-48 fragment of lambda Cro repressor. Site-specific hydrolysis by hydroxylamine was used to cleave the fusion protein, releasing the human polypeptide. The structure of the obtained hPP has been studied by scanning microcalorimetry and circular dichroism spectrometry. It has been shown that hPP in solutions close to neutral has a compact and unique spatial structure with an extended hydrophobic core. This structure is stable at 20 degrees C and co-operatively breaks down upon heating from this temperature.

Characterization and functional analysis of the Paralichthys olivaceus prdm1 gene promoter.

PubMed

Li, Peizhen; Wang, Bo; Cao, Dandan; Liu, Yuezhong; Zhang, Quanqi; Wang, Xubo

2017-10-01

PR domain containing protein 1 (Prdm1) is a transcriptional repressor identified in various species and plays multiple important roles in immune response and embryonic development. However, little is known about the transcriptional regulation of the prdm1 gene. This study aims to characterize the promoter of Paralichthys olivaceus prdm1 (Po-prdm1) gene and determine the regulatory mechanism of Po-prdm1 expression. A 2000bp-long 5'-flanking region (translation initiation site designated as +1) of the Po-prdm1 gene was isolated and characterized. The regulatory elements in this fragment were then investigated and many putative transcription factor (TF) binding sites involved in immunity and multiple tissue development were identified. A 5'-deletion analysis was then conducted, and the ability of the deletion mutants to promote luciferase and green fluorescent protein (GFP) expression in a flounder gill cell line was examined. The results revealed that the minimal promoter is located in the region between -446 and -13bp, and the region between -1415 and -13bp enhanced the promoter activity. Site-directed mutation analysis was subsequently performed on the putative regulatory elements sites, and the results indicated that FOXP1, MSX and BCL6 binding sites play negative functional roles in the regulation of the Po-prdm1 expression in FG cells. In vivo analysis demonstrated that a GFP reporter gene containing 1.4kb-long promoter fragment (-1415/-13) was expressed in the head and trunk muscle fibres of transient transgenic zebrafish embryos. Our study provided the basic information for the exploration of Po-prdm1 regulation and expression. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel principle for partial agonism of liver X receptor ligands. Competitive recruitment of activators and repressors.

PubMed

Albers, Michael; Blume, Beatrix; Schlueter, Thomas; Wright, Matthew B; Kober, Ingo; Kremoser, Claus; Deuschle, Ulrich; Koegl, Manfred

2006-02-24

Partial, selective activation of nuclear receptors is a central issue in molecular endocrinology but only partly understood. Using LXRs as an example, we show here that purely agonistic ligands can be clearly and quantitatively differentiated from partial agonists by the cofactor interactions they induce. Although a pure agonist induces a conformation that is incompatible with the binding of repressors, partial agonists such as GW3965 induce a state where the interaction not only with coactivators, but also corepressors is clearly enhanced over the unliganded state. The activities of the natural ligand 22(R)-hydroxycholesterol and of a novel quinazolinone ligand, LN6500 can be further differentiated from GW3965 and T0901317 by their weaker induction of coactivator binding. Using biochemical and cell-based assays, we show that the natural ligand of LXR is a comparably weak partial agonist. As predicted, we find that a change in the coactivator to corepressor ratio in the cell will affect NCoR recruiting compounds more dramatically than NCoR-dissociating compounds. Our data show how competitive binding of coactivators and corepressors can explain the tissue-specific behavior of partial agonists and open up new routes to a rational design of partial agonists for LXRs.

Identification and characterization of PhbF: A DNA binding protein with regulatory role in the PHB metabolism of Herbaspirillum seropedicae SmR1

PubMed Central

2011-01-01

Background Herbaspirillum seropedicae SmR1 is a nitrogen fixing endophyte associated with important agricultural crops. It produces polyhydroxybutyrate (PHB) which is stored intracellularly as granules. However, PHB metabolism and regulatory control is not yet well studied in this organism. Results In this work we describe the characterization of the PhbF protein from H. seropedicae SmR1 which was purified and characterized after expression in E. coli. The purified PhbF protein was able to bind to eleven putative promoters of genes involved in PHB metabolism in H. seropedicae SmR1. In silico analyses indicated a probable DNA-binding sequence which was shown to be protected in DNA footprinting assays using purified PhbF. Analyses using lacZ fusions showed that PhbF can act as a repressor protein controlling the expression of PHB metabolism-related genes. Conclusions Our results indicate that H. seropedicae SmR1 PhbF regulates expression of phb-related genes by acting as a transcriptional repressor. The knowledge of the PHB metabolism of this plant-associated bacterium may contribute to the understanding of the plant-colonizing process and the organism's resistance and survival in planta. PMID:21999748

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

PubMed Central

Akay, Alper; Craig, Ashley; Lehrbach, Nicolas; Larance, Mark; Pourkarimi, Ehsan; Wright, Jane E.; Lamond, Angus; Miska, Eric; Gartner, Anton

2013-01-01

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways. PMID:24258276

Identification and characterization of a nuclear localization signal of TRIM28 that overlaps with the HP1 box

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

Moriyama, Tetsuji; Sangel, Percival; Yamaguchi, Hiroki

2015-07-03

Tripartite motif-containing 28 (TRIM28) is a transcription regulator, which forms a repressor complex containing heterochromatin protein 1 (HP1). Here, we report identification of a nuclear localization signal (NLS) within the 462-494 amino acid region of TRIM28 that overlaps with its HP1 binding site, HP1 box. GST-pulldown experiments revealed the interaction of the arginine-rich TRIM28 NLS with various importin α subtypes (α1, α2 and α4). In vitro transport assay demonstrated that nuclear localization of GFP-TRIM28 NLS is mediated by importin αs, in conjunction with importin β1 and Ran. Further, we demonstrated that HP1 and importin αs compete for binding to TRIM28. Together,more » our findings suggest that importin α has an essential role in the nuclear delivery and preferential HP1 interaction of TRIM28. - Highlights: • TRIM28 contains an NLS within the 462-494 amino acid region. • The nuclear import of TRIM28 is mediated by importin α/importin β1. • TRIM28 NLS overlaps with HP1 Box. • HP1 and importin α compete for binding to TRIM28.« less

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila.

PubMed

Ji, Yingbiao; Tulin, Alexei V

2016-10-01

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates the posttranscriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germ line stem cell maintenance and oocyte localization. The mRNA targets regulated by Hrp38 are mostly unknown. We identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. We found that Hrp38 binds to the 3' untranslated region (UTR) of Nanos mRNA, which contains a translation control element. We have demonstrated that translation of the luciferase reporter bearing the Nanos 3' UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. Our data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to the Nanos 3' UTR, increasing the translation in vivo and in vitro hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. We conclude that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

Kamitani, Shinya; Ohbayashi, Norihiko; Ikeda, Osamu

Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation, and survival in immune responses, hematopoiesis, neurogenesis, and other biological processes. Recently, we showed that KAP1 is a novel STAT-binding partner that regulates STAT3-mediated transactivation. KAP1 is a universal co-repressor protein for the KRAB zinc finger protein superfamily of transcriptional repressors. In this study, we found KAP1-dependent repression of interferon (IFN)/STAT1-mediated signaling. We also demonstrated that endogenous KAP1 associates with endogenous STAT1 in vivo. Importantly, a small-interfering RNA-mediated reduction in KAP1 expression enhanced IFN-induced STAT1-dependent IRF-1 gene expression. These results indicate that KAP1 may act as an endogenous regulatormore » of the IFN/STAT1 signaling pathway.« less

SKI promotes Smad3 linker phosphorylations associated with the tumor-promoting trait of TGFbeta.

PubMed

Lin, Qiushi; Chen, Dahu; Timchenko, Nikolai A; Medrano, Estela E

2010-05-01

The transcriptional co-regulator SKI is a potent inhibitor of TGFbeta-growth inhibitory signals. SKI binds to receptor-activated Smads in the nucleus, forming repressor complexes containing HDACs, mSin3, NCoR, and other protein partners. Alternatively, SKI binds to activated Smads in the cytoplasm, preventing their nuclear translocation. SKI is necessary for anchorage-independent growth of melanoma cells in vitro, and most important, for human melanoma xenograft growth in vivo. We recently identified a novel role of SKI in TGFbeta signaling. SKI promotes the switch of Smad3 from repressor of proliferation to activator of oncogenesis by facilitating phosphorylations in the linker domain. High levels of endogenous SKI are required by the tumor promoting trait of TGFbeta to induce expression of the plasminogen-activator inhibitor-1 (PAI-1), sustained expression of C-Myc and for aborting upregulation of p21(Waf-1). Here we discuss how SKI diversifies and amplifies its functions by associating with multiple protein partners and by promoting Smad3 linker phosphorylation(s) in response to TGFbeta signaling in melanoma cells.

NRSF-dependent epigenetic mechanisms contribute to programming of stress-sensitive neurons by neonatal experience, promoting resilience.

PubMed

Singh-Taylor, A; Molet, J; Jiang, S; Korosi, A; Bolton, J L; Noam, Y; Simeone, K; Cope, J; Chen, Y; Mortazavi, A; Baram, T Z

2018-03-01

Resilience to stress-related emotional disorders is governed in part by early-life experiences. Here we demonstrate experience-dependent re-programming of stress-sensitive hypothalamic neurons, which takes place through modification of neuronal gene expression via epigenetic mechanisms. Specifically, we found that augmented maternal care reduced glutamatergic synapses onto stress-sensitive hypothalamic neurons and repressed expression of the stress-responsive gene, Crh. In hypothalamus in vitro, reduced glutamatergic neurotransmission recapitulated the repressive effects of augmented maternal care on Crh, and this required recruitment of the transcriptional repressor repressor element-1 silencing transcription factor/neuron restrictive silencing factor (NRSF). Increased NRSF binding to chromatin was accompanied by sequential repressive epigenetic changes which outlasted NRSF binding. chromatin immunoprecipitation-seq analyses of NRSF targets identified gene networks that, in addition to Crh, likely contributed to the augmented care-induced phenotype, including diminished depression-like and anxiety-like behaviors. Together, we believe these findings provide the first causal link between enriched neonatal experience, synaptic refinement and induction of epigenetic processes within specific neurons. They uncover a novel mechanistic pathway from neonatal environment to emotional resilience.

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

PubMed Central

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

1997-01-01

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

Imprinting regulator DNMT3L is a transcriptional repressor associated with histone deacetylase activity.

PubMed

Aapola, Ulla; Liiv, Ingrid; Peterson, Pärt

2002-08-15

DNMT3L is a regulator of imprint establishment of normally methylated maternal genomic sequences. DNMT3L shows high similarity to the de novo DNA methyltransferases, DNMT3A and DNMT3B, however, the amino acid residues needed for DNA cytosine methyltransferase activity have been lost from the DNMT3L protein sequence. Apart from methyltransferase activity, Dnmt3a and Dnmt3b serve as transcriptional repressors associating with histone deacetylase (HDAC) activity. Here we show that DNMT3L can also repress transcription by binding directly to HDAC1 protein. We have identified the PHD-like zinc finger of the ATRX domain as a main repression motif of DNMT3L, through which DNMT3L recruits the HDAC activity needed for transcriptional silencing. Furthermore, we show that DNMT3L protein contains an active nuclear localisation signal at amino acids 156-159. These results describe DNMT3L as a co-repressor protein and suggest that a transcriptionally repressed chromatin organisation through HDAC activity is needed for establishment of genomic imprints.

Crystal structure of enterococcus faecalis sly A-like transcriptional factor.

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

Wu, R.; Zhang, R.; Zagnitko, O.

2003-05-30

The crystal structure of a SlyA transcriptional regulator at 1.6 {angstrom} resolution is presented, and structural relationships between members of the MarR/SlyA family are discussed. The SlyA family, which includes SlyA, Rap, Hor, and RovA proteins, is widely distributed in bacterial and archaeal genomes. Current evidence suggests that SlyA-like factors act as repressors, activators, and modulators of gene transcription. These proteins have been shown to up-regulate the expression of molecular chaperones, acid-resistance proteins, and cytolysin, and down-regulate several biosynthetic enzymes. The structure of SlyA from Enterococcus faecalis, determined as a part of an ongoing structural genomics initiative (www.mcsg.anl.gov), revealed themore » same winged helix DNA-binding motif that was recently found in the MarR repressor from Escherichia coli and the MexR repressor from Pseudomonas aeruginosa, a sequence homologue of MarR. Phylogenetic analysis of the MarR/SlyA family suggests that Sly is placed between the SlyA and MarR subfamilies and shows significant sequence similarity to members of both subfamilies.« less

Phosphorylation of Trihelix Transcriptional Repressor ASR3 by MAP KINASE4 Negatively Regulates Arabidopsis Immunity

PubMed Central

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

2015-01-01

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. PMID:25770109

Analysis of the promoter of the cudA gene reveals novel mechanisms of Dictyostelium cell type differentiation.

PubMed

Fukuzawa, M; Williams, J G

2000-06-01

The cudA gene encodes a nuclear protein that is essential for normal multicellular development. At the slug stage cudA is expressed in the prespore cells and in a sub-region of the prestalk zone. We show that cap site distal promoter sequences direct cudA expression in prespore cells, while proximal sequences direct expression in the prestalk sub-region. The promoter domain that directs prespore-specific transcription consists of a positively acting region, that has the potential to direct expression in all cells within the slug, and a negatively acting region that prevents expression in the prestalk cells. Dd-STATa is the STAT protein that regulates commitment to stalk cell gene expression, where it is known to function as a transcriptional repressor. We show that Dd-STATa binds in vitro to the positively acting part of the prespore domain of the cudA promoter. However, Dd-STATa cannot be utilised for this purpose in vivo, because analysis of a Dd-STATa null mutant strain shows that Dd-STATa is not necessary for cudA transcription in prespore cells. In contrast, the part of the cudA promoter that directs prestalk-specific expression contains a binding site for Dd-STATa that is essential for its biological activity. Dd-STATa appears therefore to serve as a direct activator of cudA transcription in prestalk cells, while a protein with a DNA binding specificity highly related to that of Dd-STATa is utilised to activate cudA transcription in prespore cells.

Superrepression through Altered Corepressor-Activated Protein:Protein Interactions.

PubMed

He, Chenlu; Custer, Gregory; Wang, Jingheng; Matysiak, Silvina; Beckett, Dorothy

2018-02-20

Small molecules regulate transcription in both eukaryotes and prokaryotes by either enhancing or repressing assembly of transcription regulatory complexes. For allosteric transcription repressors, superrepressor mutants can exhibit increased sensitivity to small molecule corepressors. However, because many transcription regulatory complexes assemble in multiple steps, the superrepressor phenotype can reflect changes in any or all of the individual assembly steps. Escherichia coli biotin operon repression complex assembly, which responds to input biotin concentration, occurs via three coupled equilibria, including corepressor binding, holorepressor dimerization, and binding of the dimer to DNA. A genetic screen has yielded superrepressor mutants that repress biotin operon transcription in vivo at biotin concentrations much lower than those required by the wild type repressor. In this work, isothermal titration calorimetry and sedimentation measurements were used to determine the superrepressor biotin binding and homodimerization properties. The results indicate that, although all variants exhibit biotin binding affinities similar to that measured for BirA wt , five of the six superrepressors show altered homodimerization energetics. Molecular dynamics simulations suggest that the altered dimerization results from perturbation of an electrostatic network that contributes to allosteric activation of BirA for dimerization. Modeling of the multistep repression complex assembly for these proteins reveals that the altered sensitivity of the transcription response to biotin concentration is readily explained solely by the altered superrepressor homodimerization energetics. These results highlight how coupled equilibria enable alterations in a transcription regulatory response to input signal through an indirect mechanism.

Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type β transforming growth factor

PubMed Central

Xu, Weidong; Angelis, Konstantina; Danielpour, David; Haddad, Maher M.; Bischof, Oliver; Campisi, Judith; Stavnezer, Ed; Medrano, Estela E.

2000-01-01

The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type β transforming growth factor (TGF-β) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-β. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-β-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-β-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-β-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-β-mediated growth inhibition in a prostate-derived epithelial cell line. PMID:10811875

Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor.

PubMed

Xu, W; Angelis, K; Danielpour, D; Haddad, M M; Bischof, O; Campisi, J; Stavnezer, E; Medrano, E E

2000-05-23

The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type beta transforming growth factor (TGF-beta) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-beta. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-beta-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-beta-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-beta-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-beta-mediated growth inhibition in a prostate-derived epithelial cell line.

Purification and DNA-binding properties of the cro-type regulatory repressor protein cng encoded by the Lactobacillus plantarum phage phi g1e.

PubMed

Kakikawa, M; Ohkubo, S; Sakate, T; Sayama, M; Taketo, A; Kodaira, K

2000-05-16

The putative repressor protein Cng (10kDa on an SDS gel) for the lytic pathway of Lactobacillus plantarum phage φg1e was purified using the Escherichia coli Pt7 system, and its DNA-binding ability for the seven operator-like sequences, the GATAC-boxes (Gb1 to Gb7), was investigated in vitro. In gel-shift assays, Cng selectively bound to the DNA fragments containing the GATAC-box(es). In addition, DNase I footprinting analysis with supercoiled DNA demonstrated that Cng can specifically cover about a 25bp region centered around each of the GATAC-boxes, although two boxes, Gb4 and Gb6, were only partially protected. Moreover, protein crosslinking experiments using glutaraldehyde suggested that Cng most likely functions as a dimer. On the other hand, the binding ability of Cpg for the GATAC-boxes in supercoiled DNA was also examined under the same conditions as in Cng; unlike Cng, Cpg covered Gb4 and Gb6 completely sufficiently as well as the other five boxes. Thus, the present and previous [Kakikawa et al., Gene 215 (1998) 371-379; 242 (2000) 155-166] results indicate a possibility that the two proteins Cng and Cpg selectively bind to the GATAC-boxes that act as operators, and can decide between the lytic or lysogenic pathways through repression of the promoter activity of P(R) as well as P(L).

Hes repressors are essential regulators of hematopoietic stem cell development downstream of Notch signaling

PubMed Central

Guiu, Jordi; Shimizu, Ritsuko; D’Altri, Teresa; Fraser, Stuart T.; Hatakeyama, Jun; Bresnick, Emery H.; Kageyama, Ryoichiro; Dzierzak, Elaine; Yamamoto, Masayuki; Espinosa, Lluis

2013-01-01

Previous studies have identified Notch as a key regulator of hematopoietic stem cell (HSC) development, but the underlying downstream mechanisms remain unknown. The Notch target Hes1 is widely expressed in the aortic endothelium and hematopoietic clusters, though Hes1-deficient mice show no overt hematopoietic abnormalities. We now demonstrate that Hes is required for the development of HSC in the mouse embryo, a function previously undetected as the result of functional compensation by de novo expression of Hes5 in the aorta/gonad/mesonephros (AGM) region of Hes1 mutants. Analysis of embryos deficient for Hes1 and Hes5 reveals an intact arterial program with overproduction of nonfunctional hematopoietic precursors and total absence of HSC activity. These alterations were associated with increased expression of the hematopoietic regulators Runx1, c-myb, and the previously identified Notch target Gata2. By analyzing the Gata2 locus, we have identified functional RBPJ-binding sites, which mutation results in loss of Gata2 reporter expression in transgenic embryos, and functional Hes-binding sites, which mutation leads to specific Gata2 up-regulation in the hematopoietic precursors. Together, our findings show that Notch activation in the AGM triggers Gata2 and Hes1 transcription, and next HES-1 protein represses Gata2, creating an incoherent feed-forward loop required to restrict Gata2 expression in the emerging HSCs. PMID:23267012

Natural product (-)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1.

PubMed

Lan, Lan; Appelman, Carl; Smith, Amber R; Yu, Jia; Larsen, Sarah; Marquez, Rebecca T; Liu, Hao; Wu, Xiaoqing; Gao, Philip; Roy, Anuradha; Anbanandam, Asokan; Gowthaman, Ragul; Karanicolas, John; De Guzman, Roberto N; Rogers, Steven; Aubé, Jeffrey; Ji, Min; Cohen, Robert S; Neufeld, Kristi L; Xu, Liang

2015-08-01

Musashi-1 (MSI1) is an RNA-binding protein that acts as a translation activator or repressor of target mRNAs. The best-characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin-dependent kinase inhibitor P21(WAF-1). We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA-binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (-)-gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti-tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (-)-gossypol with the RNA binding pocket of MSI1. We further showed that (-)-gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (-)-gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (-)-gossypol as a potential small molecule inhibitor of MSI1-RNA interaction, and suggests that inhibition of MSI1's RNA binding activity may be an effective anti-cancer strategy. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells

PubMed Central

2015-01-01

Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >105 members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics. PMID:25360681

The aryl hydrocarbon receptor repressor - More than a simple feedback inhibitor of AhR signaling: Clues for its role in inflammation and cancer.

PubMed

Vogel, Christoph F A; Haarmann-Stemmann, Thomas

2017-02-01

The aryl hydrocarbon receptor repressor (AhRR) was first described as a specific competitive repressor of aryl hydrocarbon receptor (AhR) activity based on its ability to dimerize with the AhR nuclear translocator (ARNT) and through direct competition of AhR/ARNT and AhRR/ARNT complexes for binding to dioxin-responsive elements (DREs). Like AhR, AhRR belongs to the basic Helix-Loop-Helix/Per-ARNT-Sim (bHLH/PAS) protein family but lacks functional ligand-binding and transactivation domains. Transient transfection experiments with ARNT and AhRR mutants examining the inhibitory mechanism of AhRR suggested a more complex mechanism than the simple mechanism of negative feedback through sequestration of ARNT to regulate AhR signaling. Recently, AhRR has been shown to act as a tumor suppressor gene in several types of cancer cells. Furthermore, epidemiological studies have found epigenetic changes and silencing of AhRR associated with exposure to cigarette smoke and cancer development. Additional studies from our laboratories have demonstrated that AhRR represses other signaling pathways including NF-κB and is capable of regulating inflammatory responses. A better understanding of the regulatory mechanisms of AhRR in AhR signaling and adverse outcome pathways leading to deregulated inflammatory responses contributing to tumor promotion and other adverse health effects is expected from future studies. This review article summarizes the characteristics of AhRR as an inhibitor of AhR activity and highlights more recent findings pointing out the role of AhRR in inflammation and tumorigenesis.

Systematic transfer of prokaryotic sensors and circuits to mammalian cells.

PubMed

Stanton, Brynne C; Siciliano, Velia; Ghodasara, Amar; Wroblewska, Liliana; Clancy, Kevin; Trefzer, Axel C; Chesnut, Jonathan D; Weiss, Ron; Voigt, Christopher A

2014-12-19

Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >10(5) members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics.

Sp1 is a transcription repressor to stanniocalcin-1 expression in TSA-treated human colon cancer cells, HT29.

PubMed

Law, Alice Y S; Yeung, B H Y; Ching, L Y; Wong, Chris K C

2011-08-01

Our previous study demonstrated that, stanniocalcin-1 (STC1) was a target of histone deacetylase (HDAC) inhibitors and was involved in trichostatin A (TSA) induced apoptosis in the human colon cancer cells, HT29. In this study, we reported that the transcriptional factor, specificity protein 1 (Sp1) in association with retinoblastoma (Rb) repressed STC1 gene transcription in TSA-treated HT29 cells. Our data demonstrated that, a co-treatment of the cells with TSA and Sp1 inhibitor, mithramycin A (MTM) led to a marked synergistic induction of STC1 transcript levels, STC1 promoter (1 kb)-driven luciferase activity and an increase of apoptotic cell population. The knockdown of Sp1 gene expression in TSA treated cells, revealed the repressor role of Sp1 in STC1 transcription. Using a protein phosphatase inhibitor okadaic acid (OKA), an increase of Sp1 hyperphosphorylation and so a reduction of its transcriptional activity, led to a significant induction of STC1 gene expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binding on STC1 proximal promoter in TSA treated cells. The binding of Sp1 to STC1 promoter was abolished by the co-treatment of MTM or OKA in TSA-treated cells. Re-ChIP assay illustrated that Sp1-mediated inhibition of STC1 transcription was associated with the recruitment of another repressor molecule, Rb. Collectively our findings identify STC1 is a downstream target of Sp1. Copyright © 2011 Wiley-Liss, Inc.

Paracoccus denitrificans possesses two BioR homologs having a role in regulation of biotin metabolism.

PubMed

Feng, Youjun; Kumar, Ritesh; Ravcheev, Dmitry A; Zhang, Huimin

2015-08-01

Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of α-proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum α-proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR-recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR-mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC-QToF-MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR-binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid-borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA-based screening failed to demonstrate that the biotin-related metabolite is involved in BioR-DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin metabolism in P. denitrificans by two BioR proteins. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Paracoccus denitrificans possesses two BioR homologs having a role in regulation of biotin metabolism

PubMed Central

Feng, Youjun; Kumar, Ritesh; Ravcheev, Dmitry A; Zhang, Huimin

2015-01-01

Recently, we determined that BioR, the GntR family of transcription factor, acts as a repressor for biotin metabolism exclusively distributed in certain species of α-proteobacteria, including the zoonotic agent Brucella melitensis and the plant pathogen Agrobacterium tumefaciens. However, the scenario is unusual in Paracoccus denitrificans, another closely related member of the same phylum α-proteobacteria featuring with denitrification. Not only does it encode two BioR homologs Pden_1431 and Pden_2922 (designated as BioR1 and BioR2, respectively), but also has six predictive BioR-recognizable sites (the two bioR homolog each has one site, whereas the two bio operons (bioBFDAGC and bioYB) each contains two tandem BioR boxes). It raised the possibility that unexpected complexity is present in BioR-mediated biotin regulation. Here we report that this is the case. The identity of the purified BioR proteins (BioR1 and BioR2) was confirmed with LC-QToF-MS. Phylogenetic analyses combined with GC percentage raised a possibility that the bioR2 gene might be acquired by horizontal gene transfer. Gel shift assays revealed that the predicted BioR-binding sites are functional for the two BioR homologs, in much similarity to the scenario seen with the BioR site of A. tumefaciens bioBFDAZ. Using the A. tumefaciens reporter system carrying a plasmid-borne LacZ fusion, we revealed that the two homologs of P. denitrificans BioR are functional repressors for biotin metabolism. As anticipated, not only does the addition of exogenous biotin stimulate efficiently the expression of bioYB operon encoding biotin transport/uptake system BioY, but also inhibits the transcription of the bioBFDAGC operon resembling the de novo biotin synthetic pathway. EMSA-based screening failed to demonstrate that the biotin-related metabolite is involved in BioR-DNA interplay, which is consistent with our former observation with Brucella BioR. Our finding defined a complex regulatory network for biotin metabolism in P. denitrificans by two BioR proteins. PMID:26037461

Brucella BioR regulator defines a complex regulatory mechanism for bacterial biotin metabolism.

PubMed

Feng, Youjun; Xu, Jie; Zhang, Huimin; Chen, Zeliang; Srinivas, Swaminath

2013-08-01

The enzyme cofactor biotin (vitamin H or B7) is an energetically expensive molecule whose de novo biosynthesis requires 20 ATP equivalents. It seems quite likely that diverse mechanisms have evolved to tightly regulate its biosynthesis. Unlike the model regulator BirA, a bifunctional biotin protein ligase with the capability of repressing the biotin biosynthetic pathway, BioR has been recently reported by us as an alternative machinery and a new type of GntR family transcriptional factor that can repress the expression of the bioBFDAZ operon in the plant pathogen Agrobacterium tumefaciens. However, quite unusually, a closely related human pathogen, Brucella melitensis, has four putative BioR-binding sites (both bioR and bioY possess one site in the promoter region, whereas the bioBFDAZ [bio] operon contains two tandem BioR boxes). This raised the question of whether BioR mediates the complex regulatory network of biotin metabolism. Here, we report that this is the case. The B. melitensis BioR ortholog was overexpressed and purified to homogeneity, and its solution structure was found to be dimeric. Functional complementation in a bioR isogenic mutant of A. tumefaciens elucidated that Brucella BioR is a functional repressor. Electrophoretic mobility shift assays demonstrated that the four predicted BioR sites of Brucella plus the BioR site of A. tumefaciens can all interact with the Brucella BioR protein. In a reporter strain that we developed on the basis of a double mutant of A. tumefaciens (the ΔbioR ΔbioBFDA mutant), the β-galactosidase (β-Gal) activity of three plasmid-borne transcriptional fusions (bioBbme-lacZ, bioYbme-lacZ, and bioRbme-lacZ) was dramatically decreased upon overexpression of Brucella bioR. Real-time quantitative PCR analyses showed that the expression of bioBFDA and bioY is significantly elevated upon removal of bioR from B. melitensis. Together, we conclude that Brucella BioR is not only a negative autoregulator but also a repressor of expression of bioY and bio operons that separately function in biotin transport and the biosynthesis pathway.

Brucella BioR Regulator Defines a Complex Regulatory Mechanism for Bacterial Biotin Metabolism

PubMed Central

Xu, Jie; Zhang, Huimin; Srinivas, Swaminath

2013-01-01

The enzyme cofactor biotin (vitamin H or B7) is an energetically expensive molecule whose de novo biosynthesis requires 20 ATP equivalents. It seems quite likely that diverse mechanisms have evolved to tightly regulate its biosynthesis. Unlike the model regulator BirA, a bifunctional biotin protein ligase with the capability of repressing the biotin biosynthetic pathway, BioR has been recently reported by us as an alternative machinery and a new type of GntR family transcriptional factor that can repress the expression of the bioBFDAZ operon in the plant pathogen Agrobacterium tumefaciens. However, quite unusually, a closely related human pathogen, Brucella melitensis, has four putative BioR-binding sites (both bioR and bioY possess one site in the promoter region, whereas the bioBFDAZ [bio] operon contains two tandem BioR boxes). This raised the question of whether BioR mediates the complex regulatory network of biotin metabolism. Here, we report that this is the case. The B. melitensis BioR ortholog was overexpressed and purified to homogeneity, and its solution structure was found to be dimeric. Functional complementation in a bioR isogenic mutant of A. tumefaciens elucidated that Brucella BioR is a functional repressor. Electrophoretic mobility shift assays demonstrated that the four predicted BioR sites of Brucella plus the BioR site of A. tumefaciens can all interact with the Brucella BioR protein. In a reporter strain that we developed on the basis of a double mutant of A. tumefaciens (the ΔbioR ΔbioBFDA mutant), the β-galactosidase (β-Gal) activity of three plasmid-borne transcriptional fusions (bioBbme-lacZ, bioYbme-lacZ, and bioRbme-lacZ) was dramatically decreased upon overexpression of Brucella bioR. Real-time quantitative PCR analyses showed that the expression of bioBFDA and bioY is significantly elevated upon removal of bioR from B. melitensis. Together, we conclude that Brucella BioR is not only a negative autoregulator but also a repressor of expression of bioY and bio operons that separately function in biotin transport and the biosynthesis pathway. PMID:23729648

Purification of bacteriophage lambda repressor

PubMed Central

Gao, Ning; Shearwin, Keith; Mack, John; Finzi, Laura; Dunlap, David

2013-01-01

Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy “footprinting” assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops. PMID:23831434

Distinctive Roles for Amygdalar CREB in Reconsolidation and Extinction of Fear Memory

ERIC Educational Resources Information Center

Tronson, Natalie C.; Wiseman, Shari L.; Neve, Rachael L.; Nestler, Eric J.; Olausson, Peter; Taylor, Jane R.

2012-01-01

Cyclic AMP response element binding protein (CREB) plays a critical role in fear memory formation. Here we determined the role of CREB selectively within the amygdala in reconsolidation and extinction of auditory fear. Viral overexpression of the inducible cAMP early repressor (ICER) or the dominant-negative mCREB, specifically within the lateral…

The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition

PubMed Central

Battistelli, C; Cicchini, C; Santangelo, L; Tramontano, A; Grassi, L; Gonzalez, F J; de Nonno, V; Grassi, G; Amicone, L; Tripodi, M

2017-01-01

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT. PMID:27452518

The Crc global regulator inhibits the Pseudomonas putida pWW0 toluene/xylene assimilation pathway by repressing the translation of regulatory and structural genes.

PubMed

Moreno, Renata; Fonseca, Pilar; Rojo, Fernando

2010-08-06

In Pseudomonas putida, the expression of the pWW0 plasmid genes for the toluene/xylene assimilation pathway (the TOL pathway) is subject to complex regulation in response to environmental and physiological signals. This includes strong inhibition via catabolite repression, elicited by the carbon sources that the cells prefer to hydrocarbons. The Crc protein, a global regulator that controls carbon flow in pseudomonads, has an important role in this inhibition. Crc is a translational repressor that regulates the TOL genes, but how it does this has remained unknown. This study reports that Crc binds to sites located at the translation initiation regions of the mRNAs coding for XylR and XylS, two specific transcription activators of the TOL genes. Unexpectedly, eight additional Crc binding sites were found overlapping the translation initiation sites of genes coding for several enzymes of the pathway, all encoded within two polycistronic mRNAs. Evidence is provided supporting the idea that these sites are functional. This implies that Crc can differentially modulate the expression of particular genes within polycistronic mRNAs. It is proposed that Crc controls TOL genes in two ways. First, Crc inhibits the translation of the XylR and XylS regulators, thereby reducing the transcription of all TOL pathway genes. Second, Crc inhibits the translation of specific structural genes of the pathway, acting mainly on proteins involved in the first steps of toluene assimilation. This ensures a rapid inhibitory response that reduces the expression of the toluene/xylene degradation proteins when preferred carbon sources become available.

A transcription activator-like effector (TALE) induction system mediated by proteolysis.

PubMed

Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

2016-04-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

A transcription activator-like effector induction system mediated by proteolysis

PubMed Central

Copeland, Matthew F.; Politz, Mark C.; Johnson, Charles B.; Markley, Andrew L.; Pfleger, Brian F.

2016-01-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications due to their customizable DNA binding specificity. In this work we expand the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded following the induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator agnostic. PMID:26854666

Characterization of the SOS meta-regulon in the human gut microbiome.

PubMed

Cornish, Joseph P; Sanchez-Alberola, Neus; O'Neill, Patrick K; O'Keefe, Ronald; Gheba, Jameel; Erill, Ivan

2014-05-01

Data from metagenomics projects remain largely untapped for the analysis of transcriptional regulatory networks. Here, we provide proof-of-concept that metagenomic data can be effectively leveraged to analyze regulatory networks by characterizing the SOS meta-regulon in the human gut microbiome. We combine well-established in silico and in vitro techniques to mine the human gut microbiome data and determine the relative composition of the SOS network in a natural setting. Our analysis highlights the importance of translesion synthesis as a primary function of the SOS response. We predict the association of this network with three novel protein clusters involved in cell wall biogenesis, chromosome partitioning and restriction modification, and we confirm binding of the SOS response transcriptional repressor to sites in the promoter of a cell wall biogenesis enzyme, a phage integrase and a death-on-curing protein. We discuss the implications of these findings and the potential for this approach for metagenome analysis.

Establishment of inducible cAMP early repressor transgenic fibroblasts in a porcine model of human type 1 diabetes mellitus.

PubMed

Jung, Eui-Man; Kim, Yu-Kyung; Lee, Geun-Shik; Hyun, Sang-Hwan; Hwang, Woo-Suk; Jeung, Eui-Bae

2012-07-01

Diabetes mellitus is a metabolic disease caused by impaired insulin secretion from the pancreatic β cells and increased insulin resistance in peripheral tissues. Recently, the overexpression of inducible cyclic AMP (cAMP) early repressor (ICER) Iγ in rodent pancreatic β cells was found to induce insulin deficiency and glucagon overproduction similar to that found in human diabetes mellitus. ICER Iγ with only a DNA binding domain interrupts the transcriptional regulation of the cAMP responsive element-binding protein (CREB) target genes. Based on this information, we hypothesized that the overexpression of ICER Iγ, the most powerful competitor to CREB, could be useful for generating a pig model of diabetes. First, we evaluated the promoter activities of the human insulin gene for the β cell-specific overexpression of ICER Iγ in the pig pancreas. The maximum promoter activity region [-1,431 nucleotides (nt) to +1 nt, +1 = the transcriptional start site] of the insulin gene presented an activity level 3-fold higher than a promoterless construct. Second, ICER Iγ overexpression controlled by this promoter region significantly blocked the glucose-mediated insulin transcription, such as that regulated by the viral promoter in the pancreatic β cell line, MIN6. This suggests that the human insulin promoter may facilitate the overexpression of ICER Iγ in porcine pancreatic β cells. In addition, the overexpression of ICER Iγ in porcine β cells may induce human-like type 1 diabetes mellitus in pigs. In the present study, we generated transgenic fibroblasts containing ICER Iγ cDNA controlled by the human insulin promoter, as well as two screening markers, the green fluorescence protein and the neomycin resistance gene. These fibroblasts may provide a source for somatic cell nuclear transfer to generate a pig model that mimics human diabetes mellitus.

Global alterations of the transcriptional landscape during yeast growth and development in the absence of Ume6-dependent chromatin modification

PubMed Central

Lardenois, Aurélie; Becker, Emmanuelle; Walther, Thomas; Law, Michael J.; Xie, Bingning; Demougin, Philippe; Strich, Randy

2017-01-01

Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MATa/α wild-type and ume6/ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes. PMID:25957495

Global alterations of the transcriptional landscape during yeast growth and development in the absence of Ume6-dependent chromatin modification.

PubMed

Lardenois, Aurélie; Becker, Emmanuelle; Walther, Thomas; Law, Michael J; Xie, Bingning; Demougin, Philippe; Strich, Randy; Primig, Michael

2015-10-01

Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MAT a/α wild-type and ume6/ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes.

Development of a Tightly Controlled Off Switch for Saccharomyces cerevisiae Regulated by Camphor, a Low-Cost Natural Product

PubMed Central

Ikushima, Shigehito; Zhao, Yu; Boeke, Jef D.

2015-01-01

Here we describe the engineering of a distant homolog of the Tet repressor, CamR, isolated from Pseudomonas putida, that is regulated by camphor, a very inexpensive small molecule (at micromolar concentrations) for use in Saccharomyces cerevisiae. The repressor was engineered by expression from a constitutive yeast promoter, fusion to a viral activator protein cassette, and codon optimization. A suitable promoter responsive to the CamR fusion protein was engineered by embedding a P. putida operator binding sequence within an upstream activating sequence (UAS)-less CYC1 promoter from S. cerevisiae. The switch, named the Camphor-Off switch, activates expression of a reporter gene in camphor-free media and represses it with micromolar concentrations of camphor. PMID:26206350

Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant

PubMed Central

Johnson, Amanda N.; Weil, P. Anthony

2017-01-01

Repressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeast Saccharomyces cerevisiae. These include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein- and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1AS). We used Rap1AS to map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1AS variant will likely also be useful for studies of the functions of Rap1 in other biological pathways. PMID:28196871

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

PubMed

Ares, Miguel A; Fernández-Vázquez, José L; Pacheco, Sabino; Martínez-Santos, Verónica I; Jarillo-Quijada, Ma Dolores; Torres, Javier; Alcántar-Curiel, María D; González-Y-Merchand, Jorge A; De la Cruz, Miguel A

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor

PubMed Central

Ares, Miguel A.; Fernández-Vázquez, José L.; Pacheco, Sabino; Martínez-Santos, Verónica I.; Jarillo-Quijada, Ma. Dolores; Torres, Javier; Alcántar-Curiel, María D.; González-y-Merchand, Jorge A.; De la Cruz, Miguel A.

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae. PMID:28278272

Repression of myoblast proliferation and fibroblast growth factor receptor 1 promoter activity by KLF10 protein.

PubMed

Parakati, Rajini; DiMario, Joseph X

2013-05-10

FGFR1 gene expression regulates myoblast proliferation and differentiation, and its expression is controlled by Krüppel-like transcription factors. KLF10 interacts with the FGFR1 promoter, repressing its activity and cell proliferation. KLF10 represses FGFR1 promoter activity and thereby myoblast proliferation. A model of transcriptional control of chicken FGFR1 gene regulation during myogenesis is presented. Skeletal muscle development is controlled by regulation of myoblast proliferation and differentiation into muscle fibers. Growth factors such as fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate cell proliferation and differentiation in numerous tissues, including skeletal muscle. Transcriptional regulation of FGFR1 gene expression is developmentally regulated by the Sp1 transcription factor, a member of the Krüppel-like factor (KLF) family of transcriptional regulators. Here, we show that another KLF transcription factor, KLF10, also regulates myoblast proliferation and FGFR1 promoter activity. Expression of KLF10 reduced myoblast proliferation by 86%. KLF10 expression also significantly reduced FGFR1 promoter activity in myoblasts and Sp1-mediated FGFR1 promoter activity in Drosophila SL2 cells. Southwestern blot, electromobility shift, and chromatin immunoprecipitation assays demonstrated that KLF10 bound to the proximal Sp factor binding site of the FGFR1 promoter and reduced Sp1 complex formation with the FGFR1 promoter at that site. These results indicate that KLF10 is an effective repressor of myoblast proliferation and represses FGFR1 promoter activity in these cells via an Sp1 binding site.

Molecular dynamics simulation of the Escherichia coli NikR protein: equilibrium conformational fluctuations reveal interdomain allosteric communication pathways.

PubMed

Bradley, Michael J; Chivers, Peter T; Baker, Nathan A

2008-05-16

Escherichia coli NikR is a homotetrameric Ni(2+)- and DNA-binding protein that functions as a transcriptional repressor of the NikABCDE nickel permease. The protein is composed of two distinct domains. The N-terminal 50 amino acids of each chain forms part of the dimeric ribbon-helix-helix (RHH) domains, a well-studied DNA-binding fold. The 83-residue C-terminal nickel-binding domain forms an ACT (aspartokinase, chorismate mutase, and TyrA) fold and contains the tetrameric interface. In this study, we have utilized an equilibrium molecular dynamics simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains to gain insight into the effects of Ni(2+) on DNA-binding activity. The molecular simulation data were analyzed using two different correlation measures based on fluctuations in atomic position and noncovalent contacts together with a clustering algorithm to define groups of residues with similar correlation patterns for both types of correlation measure. Based on these analyses, we have defined a series of residue interrelationships that describe an allosteric communication pathway between the Ni(2+)- and DNA-binding sites, which are separated by 40 A. Several of the residues identified by our analyses have been previously shown experimentally to be important for NikR function. An additional subset of the identified residues structurally connects the experimentally implicated residues and may help coordinate the allosteric communication between the ACT and RHH domains.

Molecular dynamics simulation of the Escherichia coli NikR protein: Equilibrium conformational fluctuations reveal inter-domain allosteric communication pathways

PubMed Central

Bradley, Michael J.; Chivers, Peter T.; Baker, Nathan A.

2008-01-01

Summary E. coliNikR is a homotetrameric Ni2+- and DNA-binding protein that functions as a transcriptional repressor of the NikABCDE nickel permease. The protein is composed of 2 distinct domains. The N-terminal fifty amino acids of each chain forms part of the dimeric ribbon-helix-helix (RHH) domains, a well-studied DNA-binding fold. The eighty-three residue C-terminal nickel-binding domain forms an ACT-fold and contains the tetrameric interface. In this study, we have utilized an equilibrium molecular dynamics (MD) simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains to gain insight into the effects of Ni on DNA-binding activity. The molecular simulation data was analyzed using two different correlation measures based on fluctuations in atomic position and non-covalent contacts, together with a clustering algorithm to define groups of residues with similar correlation patterns for both types of correlation measure. Based on these analyses, we have defined a series of residue interrelationships that describe an allosteric communication pathway between the Ni2+ and DNA binding sites, which are separated by 40 Å. Several of the residues identified by our analyses have been previously shown experimentally to be important for NikR function. An additional subset of the identified residues structurally connects the experimentally implicated residues and may help coordinate the allosteric communication between the ACT and RHH domains. PMID:18433769

Modified nucleotides reveal the indirect role of the central base pairs in stabilizing the lac repressor-operator complex.

PubMed Central

Zhang, X; Gottlieb, P A

1995-01-01

Guanine residues in the lac operator were replaced by 2-aminopurine or purine analogues, pairing the modified nucleotides with C. 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, 0.1 mM DTE, pH 7.6 at 25 degrees C. These measurements revealed five positions that destabilized the complex when substituted with either analogue. Two positions, which are related by a 2-fold symmetry, are in the major groove of the operator thought to directly interact with the protein. Three sites were in the central region of the operator. A purine analogue at a sixth site perturbed the local DNA structure and destabilized the complex. Alkylation interference experiments of the 2-aminopurine substituted operators demonstrated that, of the five affected, two substitutions displayed altered phosphate interference patterns at the phosphate adjacent to the substituted base. For these operators, complex formation was measured in different concentrations of KCl to assess the contribution of counterion release to the bimolecular process. The results indicated that both complexes were similar to wild-type, although minor changes were observed. The Kobs of the complex was then measured when 2-aminopurine or purine analogues were paired with uracil nucleotide, a base pair that serves to stabilize the DNA. The introduction of the new base pairs revealed two effects on the bimolecular interaction. For those operator sites that are thought to perturb the interaction directly, the affinity of the complex was weakened to levels observed for the singly-substituted operators. In contrast, the nucleotides of 2-aminopurine paired with uracil positioned in the central region of the operator served to enhance the stability of the complex. The purine-uracil base pair substitution on the other hand had a significant destabilizing effect on the interaction. We propose that the central base pairs modulate binding of the complex by altering the intrinsic properties of the DNA. Two specific attributes are required to achieve the lowest free energy of interaction. The DNA must have two interstrand hydrogen bonds to stabilize the duplex and it must have properties associated with directional bending or unwinding. This analysis does not rule out contributions by direct interactions between the protein and the central region of the operator but underscores how indirect effects play a major role in complex formation in this system. Images PMID:7784203

DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor

NASA Astrophysics Data System (ADS)

Vonderach, Matthias; Byrne, Dominic P.; Barran, Perdita E.; Eyers, Patrick A.; Eyers, Claire E.

2018-06-01

The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKAc) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKAc- and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKAc-regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κB DNA.

Regulation of the plasma cell transcription factor Blimp-1 gene by Bach2 and Bcl6.

PubMed

Ochiai, Kyoko; Muto, Akihiko; Tanaka, Hiromu; Takahashi, Shinichiro; Igarashi, Kazuhiko

2008-03-01

B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differentiation. Prior to the terminal differentiation into plasma cells, Blimp-1 expression is suppressed in B cells by transcription repressors BTB and CNC homology 2 (Bach2) and B cell lymphoma 6 (Bcl6). Bach2 binds to the Maf recognition element (MARE) of the promoter upstream region of the Blimp-1 gene (Prdm1) by forming a heterodimer with MafK. Bach2 and Bcl6 were found to interact with each other in B cells. While both Bach2 and Bcl6 possess the BTB domain which mediates protein-protein interactions, they interacted in a BTB-independent manner. Bcl6 is known to repress Prdm1 through a Bcl6 recognition element 1 in the intron 5, in which a putative, evolutionarily conserved MARE was identified. Both repressed the expression of a reporter gene containing the intron 5 region depending on the presence of the respective binding sites in 18-81 pre-B cells. Co-expression of Bach2 and Bcl6 resulted in further repression of the reporter plasmid. Chromatin immunoprecipitation assays showed MafK to bind to the intron MARE in various B cell lines, thus suggesting that it binds as a heterodimer with Bach2. Therefore, the interaction between Bach2 and Bcl6 might be crucial for the proper repression of Prdm1 in B cells.

The mechanism and high-free-energy transition state of lac repressor–lac operator interaction

PubMed Central

Sengupta, Rituparna; Capp, Michael W.; Shkel, Irina A.

2017-01-01

Abstract Significant, otherwise-unavailable information about mechanisms and transition states (TS) of protein folding and binding is obtained from solute effects on rate constants. Here we characterize TS for lac repressor(R)–lac operator(O) binding by analyzing effects of RO-stabilizing and RO-destabilizing solutes on association (ka) and dissociation (kd) rate constants. RO-destabilizing solutes (urea, KCl) reduce ka comparably (urea) or more than (KCl) they increase kd, demonstrating that they destabilize TS relative to reactants and RO, and that TS exhibits most of the Coulombic interactions between R and O. Strikingly, three solutes which stabilize RO by favoring burial/dehydration of amide oxygens and anionic phosphate oxygens all reduce kd without affecting ka significantly. The lack of stabilization of TS by these solutes indicates that O phosphates remain hydrated in TS and that TS preferentially buries aromatic carbons and amide nitrogens while leaving amide oxygens exposed. In our proposed mechanism, DNA-binding-domains (DBD) of R insert in major grooves of O pre-TS, forming most Coulombic interactions of RO and burying aromatic carbons. Nucleation of hinge helices creates TS, burying sidechain amide nitrogens. Post-TS, hinge helices assemble and the DBD-hinge helix-O-DNA module docks on core repressor, partially dehydrating phosphate oxygens and tightening all interfaces to form RO. PMID:29036376

The Mannitol Operon Repressor MTIR belongs to a new class of transcription regulators in bacteria.

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

Tan, K.; Borovilos, M.; Zhou, M

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, representingmore » 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.« less

The RY/Sph element mediates transcriptional repression of maturation genes from late maturation to early seedling growth.

PubMed

Guerriero, Gea; Martin, Nathalie; Golovko, Anna; Sundström, Jens F; Rask, Lars; Ezcurra, Ines

2009-11-01

In orthodox seeds, the transcriptional activator ABI3 regulates two major stages in embryo maturation: a mid-maturation (MAT) stage leading to accumulation of storage compounds, and a late maturation (LEA) stage leading to quiescence and desiccation tolerance. Our aim was to elucidate mechanisms for transcriptional shutdown of MAT genes during late maturation, to better understand phase transition between MAT and LEA stages. Using transgenic and transient approaches in Nicotiana, we examined activities of two ABI3-dependent reporter genes driven by multimeric RY and abscisic acid response elements (ABREs) from a Brassica napus napin gene, termed RY and ABRE, where the RY reporter requires ABI3 DNA binding. Expression of RY peaks during mid-maturation and drops during late maturation, mimicking the MAT gene program, and in Arabidopsis thaliana RY elements are over-represented in MAT, but not in LEA, genes. The ABI3 transactivation of RY is inhibited by staurosporine, by a PP2C phosphatase, and by a repressor of maturation genes, VAL1/HSI2. The RY element mediates repression of MAT genes, and we propose that transcriptional shutdown of the MAT program during late maturation involves inhibition of ABI3 DNA binding by dephosphorylation. Later, during seedling growth, VAL1/HSI2 family repressors silence MAT genes by binding RY elements.

The Catabolite Repressor Protein-Cyclic AMP Complex Regulates csgD and Biofilm Formation in Uropathogenic Escherichia coli.

PubMed

Hufnagel, David A; Evans, Margery L; Greene, Sarah E; Pinkner, Jerome S; Hultgren, Scott J; Chapman, Matthew R

2016-12-15

The extracellular matrix protects Escherichia coli from immune cells, oxidative stress, predation, and other environmental stresses. Production of the E. coli extracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenic E. coli (UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms through csgD The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion of cyaA resulted in reduced extracellular matrix production and biofilm formation. The catabolite repressor protein (CRP) positively regulated csgD transcription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains ΔcyaA and Δcrp did not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within the csgD-csgB intergenic region, and purified CRP could gel shift the csgD-csgB intergenic region. Additionally, we found that CRP binded upstream of kpsMT, which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influence E. coli biofilms through transcriptional regulation of csgD IMPORTANCE The catabolite repressor protein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on the Escherichia coli chromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874-5893, 2004, https://dx.doi.org/10.1093/nar/gkh908). Glucose inhibits E. coli biofilm formation, and ΔcyaA and Δcrp mutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406-3410, 2002, https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms through csgD Additionally, we propose that cAMP may work as a signaling compound for uropathogenic E. coli (UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

HsfB2b-mediated repression of PRR7 directs abiotic stress responses of the circadian clock.

PubMed

Kolmos, Elsebeth; Chow, Brenda Y; Pruneda-Paz, Jose L; Kay, Steve A

2014-11-11

The circadian clock perceives environmental signals to reset to local time, but the underlying molecular mechanisms are not well understood. Here we present data revealing that a member of the heat shock factor (Hsf) family is involved in the input pathway to the plant circadian clock. Using the yeast one-hybrid approach, we isolated several Hsfs, including Heat Shock Factor B2b (HsfB2b), a transcriptional repressor that binds the promoter of Pseudo Response Regulator 7 (PRR7) at a conserved binding site. The constitutive expression of HsfB2b leads to severely reduced levels of the PRR7 transcript and late flowering and elongated hypocotyls. HsfB2b function is important during heat and salt stress because HsfB2b overexpression sustains circadian rhythms, and the hsfB2b mutant has a short circadian period under these conditions. HsfB2b is also involved in the regulation of hypocotyl growth under warm, short days. Our findings highlight the role of the circadian clock as an integrator of ambient abiotic stress signals important for the growth and fitness of plants.

An allelic series reveals essential roles for FY in plant development in addition to flowering-time control.

PubMed

Henderson, Ian R; Liu, Fuquan; Drea, Sinead; Simpson, Gordon G; Dean, Caroline

2005-08-01

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.

Ski co-repressor complexes maintain the basal repressed state of the TGF-beta target gene, SMAD7, via HDAC3 and PRMT5.

PubMed

Tabata, Takanori; Kokura, Kenji; Ten Dijke, Peter; Ishii, Shunsuke

2009-01-01

The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co-repressors and interact with other co-repressors such as N-CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c-Myb. In particular, the inhibition of TGF-beta-induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF-beta target gene, in TGF-beta-untreated HepG2 cells. Knockdown of these components using siRNA led to up-regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF-beta-responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase.

FOREVER YOUNG FLOWER Negatively Regulates Ethylene Response DNA-Binding Factors by Activating an Ethylene-Responsive Factor to Control Arabidopsis Floral Organ Senescence and Abscission1

PubMed Central

Li, Pei-Fang; Lee, Yung-I; Yang, Chang-Hsien

2015-01-01

In this study of Arabidopsis (Arabidopsis thaliana), we investigated the relationship between FOREVER YOUNG FLOWER (FYF) and Ethylene Response DNA-binding Factors (EDFs) and functionally analyzed a key FYF target, an Ethylene-Responsive Factor (ERF), that controls flower senescence/abscission. Ectopic expression of EDF1/2/3/4 caused promotion of flower senescence/abscission and the activation of the senescence-associated genes. The presence of a repressor domain in EDFs and the enhancement of the promotion of senescence/abscission in EDF1/2/3/4+SRDX (converting EDFs to strong repressors by fusion with the ERF-associated amphiphilic repression motif repression domain SRDX) transgenic plants suggested that EDFs act as repressors. The significant reduction of β-glucuronidase (GUS) expression by 35S:FYF in EDF1/2/3/4:GUS plants indicates that EDF1/2/3/4 functions downstream of FYF in regulating flower senescence/abscission. In this study, we also characterized an ERF gene, FOREVER YOUNG FLOWER UP-REGULATING FACTOR1 (FUF1), which is up-regulated by FYF during flower development. Ectopic expression of FUF1 caused similar delayed flower senescence/abscission as seen in 35S:FYF plants. This phenotype was correlated with deficient abscission zone formation, ethylene insensitivity, and down-regulation of EDF1/2/3/4 and abscission-associated genes in 35S:FUF1 flowers. In contrast, significant promotion of flower senescence/abscission and up-regulation of EDF1/2/3/4 were observed in 35S:FUF1+SRDX transgenic dominant-negative plants, in which FUF1 is converted to a potent repressor by fusion to an SRDX-suppressing motif. Thus, FUF1 acts as an activator in suppressing EDF1/2/3/4 function and senescence/abscission of the flowers. Our results reveal that FYF regulates flower senescence/abscission by negatively regulating EDF1/2/3/4, which is the downstream gene in the ethylene response, by activating FUF1 in Arabidopsis. PMID:26063506

FOREVER YOUNG FLOWER Negatively Regulates Ethylene Response DNA-Binding Factors by Activating an Ethylene-Responsive Factor to Control Arabidopsis Floral Organ Senescence and Abscission.

PubMed

Chen, Wei-Han; Li, Pei-Fang; Chen, Ming-Kun; Lee, Yung-I; Yang, Chang-Hsien

2015-08-01

In this study of Arabidopsis (Arabidopsis thaliana), we investigated the relationship between FOREVER YOUNG FLOWER (FYF) and Ethylene Response DNA-binding Factors (EDFs) and functionally analyzed a key FYF target, an Ethylene-Responsive Factor (ERF), that controls flower senescence/abscission. Ectopic expression of EDF1/2/3/4 caused promotion of flower senescence/abscission and the activation of the senescence-associated genes. The presence of a repressor domain in EDFs and the enhancement of the promotion of senescence/abscission in EDF1/2/3/4+SRDX (converting EDFs to strong repressors by fusion with the ERF-associated amphiphilic repression motif repression domain SRDX) transgenic plants suggested that EDFs act as repressors. The significant reduction of β-glucuronidase (GUS) expression by 35S:FYF in EDF1/2/3/4:GUS plants indicates that EDF1/2/3/4 functions downstream of FYF in regulating flower senescence/abscission. In this study, we also characterized an ERF gene, FOREVER YOUNG FLOWER UP-REGULATING FACTOR1 (FUF1), which is up-regulated by FYF during flower development. Ectopic expression of FUF1 caused similar delayed flower senescence/abscission as seen in 35S:FYF plants. This phenotype was correlated with deficient abscission zone formation, ethylene insensitivity, and down-regulation of EDF1/2/3/4 and abscission-associated genes in 35S:FUF1 flowers. In contrast, significant promotion of flower senescence/abscission and up-regulation of EDF1/2/3/4 were observed in 35S:FUF1+SRDX transgenic dominant-negative plants, in which FUF1 is converted to a potent repressor by fusion to an SRDX-suppressing motif. Thus, FUF1 acts as an activator in suppressing EDF1/2/3/4 function and senescence/abscission of the flowers. Our results reveal that FYF regulates flower senescence/abscission by negatively regulating EDF1/2/3/4, which is the downstream gene in the ethylene response, by activating FUF1 in Arabidopsis. © 2015 American Society of Plant Biologists. All Rights Reserved.

Arginine methylation promotes translation repression activity of eIF4G-binding protein, Scd6.

PubMed

Poornima, Gopalakrishna; Shah, Shanaya; Vignesh, Venkadasubramanian; Parker, Roy; Rajyaguru, Purusharth I

2016-11-02

Regulation of translation plays a critical role in determining mRNA fate. A new role was recently reported for a subset of RGG-motif proteins in repressing translation initiation by binding eIF4G1. However the signaling mechanism(s) that leads to spatial and temporal regulation of repression activity of RGG-motif proteins remains unknown. Here we report the role of arginine methylation in regulation of repression activity of Scd6, a conserved RGG-motif protein. We demonstrate that Scd6 gets arginine methylated at its RGG-motif and Hmt1 plays an important role in its methylation. We identify specific methylated arginine residues in the Scd6 RGG-motif in vivo We provide evidence that methylation augments Scd6 repression activity. Arginine methylation defective (AMD) mutant of Scd6 rescues the growth defect caused by overexpression of Scd6, a feature of translation repressors in general. Live-cell imaging of the AMD mutant revealed that it is defective in inducing formation of stress granules. Live-cell imaging and pull-down results indicate that it fails to bind eIF4G1 efficiently. Consistent with these results, a strain lacking Hmt1 is also defective in Scd6-eIF4G1 interaction. Our results establish that arginine methylation augments Scd6 repression activity by promoting eIF4G1-binding. We propose that arginine methylation of translation repressors with RGG-motif could be a general modulator of their repression activity. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

LEUNIG_HOMOLOG transcriptional co-repressor mediates aluminium sensitivity through PECTIN METHYLESTERASE46-modulated root cell wall pectin methylesterification in Arabidopsis.

PubMed

Geng, Xiaoyu; Horst, Walter J; Golz, John F; Lee, Joanne E; Ding, Zhaojun; Yang, Zhong-Bao

2017-05-01

A major factor determining aluminium (Al) sensitivity in higher plants is the binding of Al to root cell walls. The Al binding capacity of cell walls is closely linked to the extent of pectin methylesterification, as the presence of methyl groups attached to the pectin backbone reduces the net negative charge of this polymer and hence limits Al binding. Despite recent progress in understanding the molecular basis of Al resistance in a wide range of plants, it is not well understood how the methylation status of pectin is mediated in response to Al stress. Here we show in Arabidopsis that mutants lacking the gene LEUNIG_HOMOLOG (LUH), a member of the Groucho-like family of transcriptional co-repressor, are less sensitive to Al-mediated repression of root growth. This phenotype is correlated with increased levels of methylated pectin in the cell walls of luh roots as well as altered expression of cell wall-related genes. Among the LUH-repressed genes, PECTIN METHYLESTERASE46 (PME46) was identified as reducing Al binding to cell walls and hence alleviating Al-induced root growth inhibition by decreasing PME enzyme activity. seuss-like2 (slk2) mutants responded to Al in a similar way as luh mutants suggesting that a LUH-SLK2 complex represses the expression of PME46. The data are integrated into a model in which it is proposed that PME46 is a major inhibitor of pectin methylesterase activity within root cell walls. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA.

PubMed

Arentson, Benjamin W; Hayes, Erin L; Zhu, Weidong; Singh, Harkewal; Tanner, John J; Becker, Donald F

2016-12-01

Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ 1 -pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon-helix-helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH-RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH-RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs. © 2016 The Author(s).

Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA

PubMed Central

Arentson, Benjamin W.; Hayes, Erin L.; Zhu, Weidong; Singh, Harkewal; Tanner, John J.; Becker, Donald F.

2016-01-01

Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon–helix–helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH–RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH–RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs. PMID:27742866

Directed evolution of a synthetic phylogeny of programmable Trp repressors.

PubMed

Ellefson, Jared W; Ledbetter, Michael P; Ellington, Andrew D

2018-04-01

As synthetic regulatory programs expand in sophistication, an ever increasing number of biological components with predictable phenotypes is required. Regulators are often 'part mined' from a diverse, but uncharacterized, array of genomic sequences, often leading to idiosyncratic behavior. Here, we generate an entire synthetic phylogeny from the canonical allosteric transcription factor TrpR. Iterative rounds of positive and negative compartmentalized partnered replication (CPR) led to the exponential amplification of variants that responded with high affinity and specificity to halogenated tryptophan analogs and novel operator sites. Fourteen repressor variants were evolved with unique regulatory profiles across five operators and three ligands. The logic of individual repressors can be modularly programmed by creating heterodimeric fusions, resulting in single proteins that display logic functions, such as 'NAND'. Despite the evolutionarily limited regulatory role of TrpR, vast functional spaces exist around this highly conserved protein scaffold and can be harnessed to create synthetic regulatory programs.

Cntnap2 expression in the cerebellum of Foxp2(R552H) mice, with a mutation related to speech-language disorder.

PubMed

Fujita, Eriko; Tanabe, Yuko; Momoi, Mariko Y; Momoi, Takashi

2012-01-11

Foxp2(R552H) knock-in (KI) mice carrying a mutation related to human speech-language disorder exhibit impaired ultrasonic vocalization and poor Purkinje cell development. Foxp2 is a forkhead domain-containing transcriptional repressor that associates with its co-repressor CtBP; Foxp2(R552H) displays reduced DNA binding activity. A genetic connection between FOXP2 and CNTNAP2 has been demonstrated in vitro, but not in vivo. Here we show that Cntnap2 mRNA levels significantly increased in the cerebellum of Foxp2(R552H) KI pups, although the cerebellar population of Foxp2-positive Purkinje cells was very small. Furthermore, Cntnap2 immunofluorescence did not decrease in the poorly developed Purkinje cells of Foxp2(R552H) KI pups, although synaptophysin immunofluorescence decreased. Cntnap2 and CtBP were ubiquitously expressed, while Foxp2 co-localized with CtBP only in Purkinje cells. Taken together, these observations suggest that Foxp2 may regulate ultrasonic vocalization by associating with CtBP in Purkinje cells; Cntnap2 may be a target of this co-repressor. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Integration host factor is necessary for lysogenization of Escherichia coli by bacteriophage P2.

PubMed

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

1990-01-01

Whether infection by bacteriophage P2 results in lysogenization of the host or vegetative growth of the phage depends upon a race between transcription from the repressor promoter Pc and the early promoter Pe; transcription from these promoters is mutually exclusive, since the Pc repressor Cox is formed from the Pe transcript and the Pe repressor C from the Pc transcript. The involvement of integration host factor (IHF) in the lysogenization of Escherichia coli K12 by P2 was tested by comparing wild-type and IHF-deficient (himA and himD) mutants. No lysogenic clones were formed following infection of the mutant bacteria. A switch plasmid that contains Pc-C-cat and Pe-cox-kan was used to test the choice for expression of Pc versus Pe. In the wild-type K12 bacteria, 20% of the clones expressed Pe transcription and 80% Pc transcription, whereas all transformed IHF-defective clones expressed transcription from Pe only. The effects of IHF on the in vivo expression of the Pe and Pc promoters were only marginal. The IHF protein was found to bind upstream of the Pe promoter, where a potential ihf sequence is located.

Molecular dynamics simulation of trp-repressor/operator complex: analysis of hydrogen bond patterns of protein DNA interaction

NASA Astrophysics Data System (ADS)

Suenaga, A.; Yatsu, C.; Komeiji, Y.; Uebayasi, M.; Meguro, T.; Yamato, I.

2000-08-01

Molecular dynamics simulation of Escherichia colitrp-repressor/operator complex was performed to elucidate protein-DNA interactions in solution for 800 ps on special-purpose computer MD-GRAPE. The Ewald summation method was employed to treat the electrostatic interaction without cutoff. DNA kept stable conformation in comparison with the result of the conventional cutoff method. Thus, the trajectories obtained were used to analyze the protein-DNA interaction and to understand the role of dynamics of water molecules forming sequence specific recognition interface. The dynamical cross-correlation map showed a significant positive correlation between the helix-turn-helix DNA-binding motifs and the major grooves of operator DNA. The extensive contact surface was stable during the simulation. Most of the contacts consisted of direct interactions between phosphates of DNA and the protein, but several water-mediated polar contacts were also observed. These water-mediated interactions, which were also seen in the crystal structure (Z. Otwinowski, et al., Nature, 335 (1998) 321) emerged spontaneously from the randomized initial configuration of the solvent. This result suggests the importance of the water-mediated interaction in specific recognition of DNA by the trp-repressor, consistent with X-ray structural information.

Functional domains of the Drosophila Engrailed protein.

PubMed Central

Han, K; Manley, J L

1993-01-01

We have studied the transcriptional activity of the Drosophila homeodomain protein Engrailed (En) by using a transient expression assay employing Schneider L2 cells. En was found to very strongly repress promoters activated by a variety of different activator proteins. However, unlike another Drosophila homeodomain-containing repressor, Even-skipped (Eve), En was unable to repress the activity of several basal promoters in the absence of activator expression. These findings indicate that En is a specific repressor of activated transcription, and suggest that En may repress transcription by a different mechanism than Eve, perhaps by interfering with interactions between transcriptional activators and the general transcription machinery. By analyzing the properties of a variety of En mutants, we identified a minimal repression domain composed of 55 residues, which can function when fused to a heterologous DNA binding domain. Like repression domains identified in the Drosophila repressors Eve and Krüppel, the En repression domain is rich in alanine residues (26%), but unlike these other domains, is moderately charged (six arginine and three glutamic acid residues). Separate regions of En that may in some circumstances function in transcriptional activation were also identified. Images PMID:8334991

Modeling the Lac repressor-operator assembly: The influence of DNA looping on Lac repressor conformation

PubMed Central

Swigon, David; Coleman, Bernard D.; Olson, Wilma K.

2006-01-01

Repression of transcription of the Escherichia coli Lac operon by the Lac repressor (LacR) is accompanied by the simultaneous binding of LacR to two operators and the formation of a DNA loop. A recently developed theory of sequence-dependent DNA elasticity enables one to relate the fine structure of the LacR–DNA complex to a wide range of heretofore-unconnected experimental observations. Here, that theory is used to calculate the configuration and free energy of the DNA loop as a function of its length and base-pair sequence, its linking number, and the end conditions imposed by the LacR tetramer. The tetramer can assume two types of conformations. Whereas a rigid V-shaped structure is observed in the crystal, EM images show extended forms in which two dimer subunits are flexibly joined. Upon comparing our computed loop configurations with published experimental observations of permanganate sensitivities, DNase I cutting patterns, and loop stabilities, we conclude that linear DNA segments of short-to-medium chain length (50–180 bp) give rise to loops with the extended form of LacR and that loops formed within negatively supercoiled plasmids induce the V-shaped structure. PMID:16785444

Mecp2 deficiency leads to altered Htr2c pre-mRNA editing and HTR2C isoform distribution in mouse hippocampus and cerebellum

USDA-ARS?s Scientific Manuscript database

Rett Syndrome (RTT) is a neurodevelopmental disorder caused by mutations in MECP2, a methyl-CpG binding protein and transcriptional repressor. CpG methylation plays an important role in genomic imprinting since imprinted genes are regulated by regions of differentially methylated CpGs (or ICs). A ...

Identification of pathways directly regulated by SHORT VEGETATIVE PHASE during vegetative and reproductive development in Arabidopsis

PubMed Central

2013-01-01

Background MADS-domain transcription factors play important roles during plant development. The Arabidopsis MADS-box gene SHORT VEGETATIVE PHASE (SVP) is a key regulator of two developmental phases. It functions as a repressor of the floral transition during the vegetative phase and later it contributes to the specification of floral meristems. How these distinct activities are conferred by a single transcription factor is unclear, but interactions with other MADS domain proteins which specify binding to different genomic regions is likely one mechanism. Results To compare the genome-wide DNA binding profile of SVP during vegetative and reproductive development we performed ChIP-seq analyses. These ChIP-seq data were combined with tiling array expression analysis, induction experiments and qRT-PCR to identify biologically relevant binding sites. In addition, we compared genome-wide target genes of SVP with those published for the MADS domain transcription factors FLC and AP1, which interact with SVP during the vegetative and reproductive phases, respectively. Conclusions Our analyses resulted in the identification of pathways that are regulated by SVP including those controlling meristem development during vegetative growth and flower development whereas floral transition pathways and hormonal signaling were regulated predominantly during the vegetative phase. Thus, SVP regulates many developmental pathways, some of which are common to both of its developmental roles whereas others are specific to only one of them. PMID:23759218

Genetics and Diet Regulate Vitamin A Production via the Homeobox Transcription Factor ISX*

PubMed Central

Lobo, Glenn P.; Amengual, Jaume; Baus, Diane; Shivdasani, Ramesh A.; Taylor, Derek; von Lintig, Johannes

2013-01-01

Low dietary intake of β-carotene is associated with chronic disease and vitamin A deficiency. β-Carotene is converted to vitamin A in the intestine by the enzyme β-carotene-15,15′-monoxygenase (BCMO1) to support vision, reproduction, immune function, and cell differentiation. Considerable variability for this key step in vitamin A metabolism, as reported in the human population, could be related to genetics and individual vitamin A status, but it is unclear how these factors influence β-carotene metabolism and vitamin A homeostasis. Here we show that the intestine-specific transcription factor ISX binds to the Bcmo1 promoter. Moreover, upon induction by the β-carotene derivative retinoic acid, this ISX binding decreased expression of a luciferase reporter gene in human colonic CaCo-2 cells indicating that ISX acts as a transcriptional repressor of BCMO1 expression. Mice deficient for this transcription factor displayed increased intestinal BCMO1 expression and produced significantly higher amounts of vitamin A from supplemental β-carotene. The ISX binding site in the human BCMO1 promoter contains a common single nucleotide polymorphism that is associated with decreased conversion rates and increased fasting blood levels of β-carotene. Thus, our study establishes ISX as a critical regulator of vitamin A production and provides a mechanistic explanation for how both genetics and diet can affect this process. PMID:23393141

MARs and MARBPs: key modulators of gene regulation and disease manifestation.

PubMed

Chattopadhyay, Samit; Pavithra, Lakshminarasimhan

2007-01-01

The DNA in eukaryotic genome is compartmentalized into various domains by a series of loops tethered onto the base of nuclear matrix. Scaffold/Matrix attachment regions (S/MAR) punctuate these attachment sites and govern the nuclear architecture by establishing chromatin boundaries. In this context, specific proteins that interact with and bind to MAR sequences called MAR binding proteins (MARBPs), are of paramount importance, as these sequences spool the proteins that regulate transcription, replication, repair and recombination. Recent evidences also suggest a role for these cis-acting elements in viral integration, replication and transcription, thereby affecting host immune system. Owing to the complex nature of these nucleotide sequences, less is known about the MARBPs that bind to and bring about diverse effects on chromatin architecture and gene function. Several MARBPs have been identified and characterized so far and the list is growing. The fact that most the MARBPs exist in a co-repressor/co-activator complex and bring about gene regulation makes them quintessential for cellular processes. This participation in gene regulation means that any perturbation in the regulation and levels of MARBPs could lead to disease conditions, particularly those caused by abnormal cell proliferation, like cancer. In the present chapter, we discuss the role of MARs and MARBPs in eukaryotic gene regulation, recombination, transcription and viral integration by altering the local chromatin structure and their dysregulation in disease manifestation

Concomitant expression of far upstream element (FUSE) binding protein (FBP) interacting repressor (FIR) and its splice variants induce migration and invasion of non-small cell lung cancer (NSCLC) cells.

PubMed

Müller, Benedikt; Bovet, Michael; Yin, Yi; Stichel, Damian; Malz, Mona; González-Vallinas, Margarita; Middleton, Alistair; Ehemann, Volker; Schmitt, Jennifer; Muley, Thomas; Meister, Michael; Herpel, Esther; Singer, Stephan; Warth, Arne; Schirmacher, Peter; Drasdo, Dirk; Matthäus, Franziska; Breuhahn, Kai

2015-11-01

Transcription factors integrate a variety of oncogenic input information, facilitate tumour growth and cell dissemination, and therefore represent promising therapeutic target structures. Because over-expression of DNA-interacting far upstream element binding protein (FBP) supports non-small cell lung cancer (NSCLC) migration, we asked whether its repressor, FBP-interacting repressor (FIR) is functionally inactivated and how FIR might affect NSCLC cell biology. Different FIR splice variants were highly expressed in the majority of NSCLCs, with the highest levels in tumours carrying genomic gains of chromosome 8q24.3, which contained the FIR gene locus. Nuclear FIR expression was significantly enriched at the invasion front of primary NSCLCs, but this did not correlate with tumour cell proliferation. FIR accumulation was associated with worse patient survival and tumour recurrence; in addition, FIR over-expression significantly correlated with lymph node metastasis in squamous cell carcinomas (SCCs). In vitro, we applied newly developed methods and modelling approaches for the quantitative and time-resolved description of the pro-migratory and pro-invasive capacities of SCC cells. siRNA-mediated silencing of all FIR variants significantly reduced the speed and directional movement of tumour cells in all phases of migration. Furthermore, sprouting efficiency and single cell invasiveness were diminished following FIR inhibition. Interestingly, the silencing of FIR isoforms lacking exon 2 (FIR(Δexon2)) alone was sufficient to reduce lateral migration and invasion. In summary, by using scale-spanning data derived from primary human tissues, quantitative cellular analyses and mathematical modelling, we have demonstrated that concomitant over-expression of FIR and its splice variants drives NSCLC migration and dissemination. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Experimental identification of specificity determinants in the domain linker of a LacI/GalR protein: bioinformatics-based predictions generate true positives and false negatives.

PubMed

Meinhardt, Sarah; Swint-Kruse, Liskin

2008-12-01

In protein families, conserved residues often contribute to a common general function, such as DNA-binding. However, unique attributes for each homolog (e.g. recognition of alternative DNA sequences) must arise from variation in other functionally-important positions. The locations of these "specificity determinant" positions are obscured amongst the background of varied residues that do not make significant contributions to either structure or function. To isolate specificity determinants, a number of bioinformatics algorithms have been developed. When applied to the LacI/GalR family of transcription regulators, several specificity determinants are predicted in the 18 amino acids that link the DNA-binding and regulatory domains. However, results from alternative algorithms are only in partial agreement with each other. Here, we experimentally evaluate these predictions using an engineered repressor comprising the LacI DNA-binding domain, the LacI linker, and the GalR regulatory domain (LLhG). "Wild-type" LLhG has altered DNA specificity and weaker lacO(1) repression compared to LacI or a similar LacI:PurR chimera. Next, predictions of linker specificity determinants were tested, using amino acid substitution and in vivo repression assays to assess functional change. In LLhG, all predicted sites are specificity determinants, as well as three sites not predicted by any algorithm. Strategies are suggested for diminishing the number of false negative predictions. Finally, individual substitutions at LLhG specificity determinants exhibited a broad range of functional changes that are not predicted by bioinformatics algorithms. Results suggest that some variants have altered affinity for DNA, some have altered allosteric response, and some appear to have changed specificity for alternative DNA ligands.

Analysis of URI nuclear interaction with RPB5 and components of the R2TP/prefoldin-like complex.

PubMed

Mita, Paolo; Savas, Jeffrey N; Ha, Susan; Djouder, Nabil; Yates, John R; Logan, Susan K

2013-01-01

Unconventional prefoldin RPB5 Interactor (URI) was identified as a transcriptional repressor that binds RNA polymerase II (pol II) through interaction with the RPB5/POLR2E subunit. Despite the fact that many other proteins involved in transcription regulation have been shown to interact with URI, its nuclear function still remains elusive. Previous mass spectrometry analyses reported that URI is part of a novel protein complex called R2TP/prefoldin-like complex responsible for the cytoplasmic assembly of RNA polymerase II. We performed a mass spectrometry (MS)-based proteomic analysis to identify nuclear proteins interacting with URI in prostate cells. We identified all the components of the R2TP/prefoldin-like complex as nuclear URI interactors and we showed that URI binds and regulates RPB5 protein stability and transcription. Moreover, we validated the interaction of URI to the P53 and DNA damage-Regulated Gene 1 (PDRG1) and show that PDRG1 protein is also stabilized by URI binding. We present data demonstrating that URI nuclear/cytoplasmic shuttling is affected by compounds that stall pol II on the DNA (α-amanitin and actinomycin-D) and by leptomycin B, an inhibitor of the CRM1 exportin that mediates the nuclear export of pol II subunits. These data suggest that URI, and probably the entire R2TP/prefoldin-like complex is exported from the nucleus through CRM1. Finally we identified putative URI sites of phosphorylation and acetylation and confirmed URI sites of post-transcriptional modification identified in previous large-scale analyses the importance of which is largely unknown. However URI post-transcriptional modification was shown to be essential for URI function and therefore characterization of novel sites of URI modification will be important to the understanding of URI function.

Analysis of URI Nuclear Interaction with RPB5 and Components of the R2TP/Prefoldin-Like Complex

PubMed Central

Mita, Paolo; Savas, Jeffrey N.; Ha, Susan; Djouder, Nabil; Yates, John R.; Logan, Susan K.

2013-01-01

Unconventional prefoldin RPB5 Interactor (URI) was identified as a transcriptional repressor that binds RNA polymerase II (pol II) through interaction with the RPB5/POLR2E subunit. Despite the fact that many other proteins involved in transcription regulation have been shown to interact with URI, its nuclear function still remains elusive. Previous mass spectrometry analyses reported that URI is part of a novel protein complex called R2TP/prefoldin-like complex responsible for the cytoplasmic assembly of RNA polymerase II. We performed a mass spectrometry (MS)-based proteomic analysis to identify nuclear proteins interacting with URI in prostate cells. We identified all the components of the R2TP/prefoldin-like complex as nuclear URI interactors and we showed that URI binds and regulates RPB5 protein stability and transcription. Moreover, we validated the interaction of URI to the P53 and DNA damage-Regulated Gene 1 (PDRG1) and show that PDRG1 protein is also stabilized by URI binding. We present data demonstrating that URI nuclear/cytoplasmic shuttling is affected by compounds that stall pol II on the DNA (α-amanitin and actinomycin-D) and by leptomycin B, an inhibitor of the CRM1 exportin that mediates the nuclear export of pol II subunits. These data suggest that URI, and probably the entire R2TP/prefoldin-like complex is exported from the nucleus through CRM1. Finally we identified putative URI sites of phosphorylation and acetylation and confirmed URI sites of post-transcriptional modification identified in previous large-scale analyses the importance of which is largely unknown. However URI post-transcriptional modification was shown to be essential for URI function and therefore characterization of novel sites of URI modification will be important to the understanding of URI function. PMID:23667685

λ-Repressor Oligomerization Kinetics at High Concentrations Using Fluorescence Correlation Spectroscopy in Zero-Mode Waveguides

PubMed Central

Samiee, K. T.; Foquet, M.; Guo, L.; Cox, E. C.; Craighead, H. G.

2005-01-01

Fluorescence correlation spectroscopy (FCS) has demonstrated its utility for measuring transport properties and kinetics at low fluorophore concentrations. In this article, we demonstrate that simple optical nanostructures, known as zero-mode waveguides, can be used to significantly reduce the FCS observation volume. This, in turn, allows FCS to be applied to solutions with significantly higher fluorophore concentrations. We derive an empirical FCS model accounting for one-dimensional diffusion in a finite tube with a simple exponential observation profile. This technique is used to measure the oligomerization of the bacteriophage λ repressor protein at micromolar concentrations. The results agree with previous studies utilizing conventional techniques. Additionally, we demonstrate that the zero-mode waveguides can be used to assay biological activity by measuring changes in diffusion constant as a result of ligand binding. PMID:15613638

Modeling of the structure and interactions of the B. anthracis antitoxin, MoxX: deletion mutant studies highlight its modular structure and repressor function

NASA Astrophysics Data System (ADS)

Chopra, Nikita; Agarwal, Shivangi; Verma, Shashikala; Bhatnagar, Sonika; Bhatnagar, Rakesh

2011-03-01

Our previous report on Bacillus anthracis toxin-antitoxin module (MoxXT) identified it to be a two component system wherein, PemK-like toxin (MoxT) functions as a ribonuclease (Agarwal S et al. JBC 285:7254-7270, 2010). The labile antitoxin (MoxX) can bind to/neutralize the action of the toxin and is also a DNA-binding protein mediating autoregulation. In this study, molecular modeling of MoxX in its biologically active dimeric form was done. It was found that it contains a conserved Ribbon-Helix-Helix (RHH) motif, consistent with its DNA-binding function. The modeled MoxX monomers dimerize to form a two-stranded antiparallel ribbon, while the C-terminal region adopts an extended conformation. Knowledge guided protein-protein docking, molecular dynamics simulation, and energy minimization was performed to obtain the structure of the MoxXT complex, which was exploited for the de novo design of a peptide capable of binding to MoxT. It was found that the designed peptide caused a decrease in MoxX binding to MoxT by 42% at a concentration of 2 μM in vitro. We also show that MoxX mediates negative transcriptional autoregulation by binding to its own upstream DNA. The interacting regions of both MoxX and DNA were identified in order to model their complex. The repressor activity of MoxX was found to be mediated by the 16 N-terminal residues that contains the ribbon of the RHH motif. Based on homology with other RHH proteins and deletion mutant studies, we propose a model of the MoxX-DNA interaction, with the antiparallel β-sheet of the MoxX dimer inserted into the major groove of its cognate DNA. The structure of the complex of MoxX with MoxT and its own upstream regulatory region will facilitate design of molecules that can disrupt these interactions, a strategy for development of novel antibacterials.

Regulated expression of the Ras effector Rin1 in forebrain neurons

PubMed Central

Dzudzor, Bartholomew; Huynh, Lucia; Thai, Minh; Bliss, Joanne M.; Nagaoka, Yoshiko; Wang, Ying; Ch'ng, Toh Hean; Jiang, Meisheng; Martin, Kelsey C.; Colicelli, John

2009-01-01

The Ras effector Rin1 is induced concomitant with synaptogenesis in forebrain neurons, where it inhibits fear conditioning and amygdala LTP. In epithelial cells, lower levels of Rin1 orchestrate receptor endocytosis. A 945bp Rin1 promoter fragment was active in hippocampal neurons and directed accurate tissue-specific and temporal expression in transgenic mice. Regulated expression in neurons and epithelial cells was mediated in part by Snail transcriptional repressors: mutation of a conserved Snail site increased expression and endogenous Snai1 was detected at the Rin1 promoter. We also describe an element closely related to, but distinct from, the consensus site for REST, a master repressor of neuronal genes. Conversion to a consensus REST sequence reduced expression in both cell types. These results provide insight into regulated expression of a neuronal Ras effector, define a promoter useful in telencephalic neuron studies, and describe a novel REST site variant directing expression to mature neurons. PMID:19837165

Interactions of the Metalloregulatory Protein SloR from Streptococcus mutans with Its Metal Ion Effectors and DNA Binding Site

PubMed Central

Corbett, John; Cornacchione, Louis; Daly, William; Galan, Diego; Wysota, Michael; Tivnan, Patrick; Collins, Justin; Nye, Dillon; Levitz, Talya; Breyer, Wendy A.; Glasfeld, Arthur

2015-01-01

ABSTRACT Streptococcus mutans is the causative agent of dental caries, a significant concern for human health, and therefore an attractive target for therapeutics development. Previous work in our laboratory has identified a homodimeric, manganese-dependent repressor protein, SloR, as an important regulator of cariogenesis and has used site-directed mutagenesis to map functions to specific regions of the protein. Here we extend those studies to better understand the structural interaction between SloR and its operator and its effector metal ions. The results of DNase I assays indicate that SloR protects a 42-bp region of DNA that overlaps the sloABC promoter on the S. mutans UA159 chromosome, while electrophoretic mobility shift and solution binding assays indicate that each of two SloR dimers binds to this region. Real-time semiquantitative reverse transcriptase PCR (real-time semi-qRT-PCR) experiments were used to determine the individual base pairs that contribute to SloR-DNA binding specificity. Solution studies indicate that Mn2+ is better than Zn2+ at specifically activating SloR to bind DNA, and yet the 2.8-Å resolved crystal structure of SloR bound to Zn2+ provides insight into the means by which selective activation by Mn2+ may be achieved and into how SloR may form specific interactions with its operator. Taken together, these experimental observations are significant because they can inform rational drug design aimed at alleviating and/or preventing S. mutans-induced caries formation. IMPORTANCE This report focuses on investigating the SloR protein as a regulator of essential metal ion transport and virulence gene expression in the oral pathogen Streptococcus mutans and on revealing the details of SloR binding to its metal ion effectors and binding to DNA that together facilitate this expression. We used molecular and biochemical approaches to characterize the interaction of SloR with Mn2+ and with its SloR recognition element to gain a clearer picture of the regulatory networks that optimize SloR-mediated metal ion homeostasis and virulence gene expression in S. mutans. These experiments can have a significant impact on caries treatment and/or prevention by revealing the S. mutans SloR-DNA binding interface as an appropriate target for the development of novel therapeutic interventions. PMID:26350131

Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

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

Storrs, Richard Wood

1992-08-01

Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by 31P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis,more » syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 Å of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an α-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.« less

Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

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

Storrs, R.W.

1992-08-01

Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by [sup 31]P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal ofmore » cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 [Angstrom] of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an [alpha]-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.« less

The human RNA-binding protein and E3 ligase MEX-3C binds the MEX-3-recognition element (MRE) motif with high affinity.

PubMed

Yang, Lingna; Wang, Chongyuan; Li, Fudong; Zhang, Jiahai; Nayab, Anam; Wu, Jihui; Shi, Yunyu; Gong, Qingguo

2017-09-29

MEX-3 is a K-homology (KH) domain-containing RNA-binding protein first identified as a translational repressor in Caenorhabditis elegans , and its four orthologs (MEX-3A-D) in human and mouse were subsequently found to have E3 ubiquitin ligase activity mediated by a RING domain and critical for RNA degradation. Current evidence implicates human MEX-3C in many essential biological processes and suggests a strong connection with immune diseases and carcinogenesis. The highly conserved dual KH domains in MEX-3 proteins enable RNA binding and are essential for the recognition of the 3'-UTR and post-transcriptional regulation of MEX-3 target transcripts. However, the molecular mechanisms of translational repression and the consensus RNA sequence recognized by the MEX-3C KH domain are unknown. Here, using X-ray crystallography and isothermal titration calorimetry, we investigated the RNA-binding activity and selectivity of human MEX-3C dual KH domains. Our high-resolution crystal structures of individual KH domains complexed with a noncanonical U-rich and a GA-rich RNA sequence revealed that the KH1/2 domains of human MEX-3C bound MRE10, a 10-mer RNA (5'-CAGAGUUUAG-3') consisting of an eight-nucleotide MEX-3-recognition element (MRE) motif, with high affinity. Of note, we also identified a consensus RNA motif recognized by human MEX-3C. The potential RNA-binding sites in the 3'-UTR of the human leukocyte antigen serotype ( HLA-A2 ) mRNA were mapped with this RNA-binding motif and further confirmed by fluorescence polarization. The binding motif identified here will provide valuable information for future investigations of the functional pathways controlled by human MEX-3C and for predicting potential mRNAs regulated by this enzyme. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Long-Range Control of Gene Expression: Emerging Mechanisms and Disruption in Disease

PubMed Central

Kleinjan, Dirk A.; van Heyningen, Veronica

2005-01-01

Transcriptional control is a major mechanism for regulating gene expression. The complex machinery required to effect this control is still emerging from functional and evolutionary analysis of genomic architecture. In addition to the promoter, many other regulatory elements are required for spatiotemporally and quantitatively correct gene expression. Enhancer and repressor elements may reside in introns or up- and downstream of the transcription unit. For some genes with highly complex expression patterns—often those that function as key developmental control genes—the cis-regulatory domain can extend long distances outside the transcription unit. Some of the earliest hints of this came from disease-associated chromosomal breaks positioned well outside the relevant gene. With the availability of wide-ranging genome sequence comparisons, strong conservation of many noncoding regions became obvious. Functional studies have shown many of these conserved sites to be transcriptional regulatory elements that sometimes reside inside unrelated neighboring genes. Such sequence-conserved elements generally harbor sites for tissue-specific DNA-binding proteins. Developmentally variable chromatin conformation can control protein access to these sites and can regulate transcription. Disruption of these finely tuned mechanisms can cause disease. Some regulatory element mutations will be associated with phenotypes distinct from any identified for coding-region mutations. PMID:15549674

Sperm and Spermatids Contain Different Proteins and Bind Distinct Egg Factors

PubMed Central

Teperek, Marta; Miyamoto, Kei; Simeone, Angela; Feret, Renata; Deery, Michael J.; Gurdon, John B.; Jullien, Jerome

2014-01-01

Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development. PMID:25244019

Molecular dynamics studies of a DNA-binding protein: 2. An evaluation of implicit and explicit solvent models for the molecular dynamics simulation of the Escherichia coli trp repressor.

PubMed Central

Guenot, J.; Kollman, P. A.

1992-01-01

Although aqueous simulations with periodic boundary conditions more accurately describe protein dynamics than in vacuo simulations, these are computationally intensive for most proteins. Trp repressor dynamic simulations with a small water shell surrounding the starting model yield protein trajectories that are markedly improved over gas phase, yet computationally efficient. Explicit water in molecular dynamics simulations maintains surface exposure of protein hydrophilic atoms and burial of hydrophobic atoms by opposing the otherwise asymmetric protein-protein forces. This properly orients protein surface side chains, reduces protein fluctuations, and lowers the overall root mean square deviation from the crystal structure. For simulations with crystallographic waters only, a linear or sigmoidal distance-dependent dielectric yields a much better trajectory than does a constant dielectric model. As more water is added to the starting model, the differences between using distance-dependent and constant dielectric models becomes smaller, although the linear distance-dependent dielectric yields an average structure closer to the crystal structure than does a constant dielectric model. Multiplicative constants greater than one, for the linear distance-dependent dielectric simulations, produced trajectories that are progressively worse in describing trp repressor dynamics. Simulations of bovine pancreatic trypsin were used to ensure that the trp repressor results were not protein dependent and to explore the effect of the nonbonded cutoff on the distance-dependent and constant dielectric simulation models. The nonbonded cutoff markedly affected the constant but not distance-dependent dielectric bovine pancreatic trypsin inhibitor simulations. As with trp repressor, the distance-dependent dielectric model with a shell of water surrounding the protein produced a trajectory in better agreement with the crystal structure than a constant dielectric model, and the physical properties of the trajectory average structure, both with and without a nonbonded cutoff, were comparable. PMID:1304396

Structural basis of JAZ repression of MYC transcription factors in jasmonate signalling

DOE PAGES

Zhang, Feng; Yao, Jian; Ke, Jiyuan; ...

2015-08-10

The plant hormone jasmonate plays crucial roles in regulating plant responses to herbivorous insects and microbial pathogens and is an important regulator of plant growth and development. Key mediators of jasmonate signalling include MYC transcription factors, which are repressed by jasmonate ZIM-domain (JAZ) transcriptional repressors in the resting state. In the presence of active jasmonate, JAZ proteins function as jasmonate co-receptors by forming a hormone-dependent complex with COI1, the F-box subunit of an SCF-type ubiquitin E3 ligase. The hormone-dependent formation of the COI1–JAZ co-receptor complex leads to ubiquitination and proteasome-dependent degradation of JAZ repressors and release of MYC proteins frommore » transcriptional repression. The mechanism by which JAZ proteins repress MYC transcription factors and how JAZ proteins switch between the repressor function in the absence of hormone and the co-receptor function in the presence of hormone remain enigmatic. In this paper, we show that Arabidopsis MYC3 undergoes pronounced conformational changes when bound to the conserved Jas motif of the JAZ9 repressor. The Jas motif, previously shown to bind to hormone as a partly unwound helix, forms a complete α-helix that displaces the amino (N)-terminal helix of MYC3 and becomes an integral part of the MYC N-terminal fold. In this position, the Jas helix competitively inhibits MYC3 interaction with the MED25 subunit of the transcriptional Mediator complex. Finally, our structural and functional studies elucidate a dynamic molecular switch mechanism that governs the repression and activation of a major plant hormone pathway.« less

Backbone dynamics in an intramolecular prolylpeptide-SH3 complex from the diphtheria toxin repressor, DtxR

PubMed Central

Bhattacharya, Nilakshee; Yi, Myunggi; Zhou, Huan-Xiang; Logan, Timothy M.

2008-01-01

Summary The diphtheria toxin repressor contains an SH3-like domain that forms an intramolecular complex with a proline-rich (Pr) peptide segment and stabilizes the inactive state of the repressor. Upon activation of DtxR by transition metals, this intramolecular complex must dissociate as the SH3 domain and Pr segment form different interactions in the active repressor. In this study we investigate the dynamics of this intramolecular complex using backbone amide nuclear spin relaxation rates determined using NMR spectroscopy and molecular dynamics trajectories. The SH3 domain in the unbound and bound states showed typical dynamics in that the secondary structures were fairly ordered with high generalized order parameters and low effective correlation times while residues in the loops connecting β-strands exhibited reduced generalized order parameters and required additional motional terms to adequately model the relaxation rates. Residues forming the Pr segment exhibited low order parameters with internal rotational correlation times on the order of 0.6 – 1 ns. Further analysis showed that the SH3 domain was rich in millisecond timescale motions while the Pr segment was rich in motions on the 100s μs timescale. Molecular dynamics simultations indicated structural rearrangements that may contribute to the observed relaxation rates and, together with the observed relaxation rate data, suggested that the Pr segment exhibits a binding ↔ unbinding equilibrium. The results of this study provide new insights into the nature of the intramolecular complex and provide a better understanding of the biological role of the SH3 domain in regulating DtxR activity. PMID:17976643

PPARγ and NF-κB regulate the gene promoter activity of their shared repressor, TNIP1

PubMed Central

Gurevich, Igor; Zhang, Carmen; Encarnacao, Priscilla C.; Struzynski, Charles P.; Livings, Sarah E.; Aneskievich, Brian J.

2011-01-01

Human TNFAIP3 interacting protein 1 (TNIP1) has diverse functions including support of HIV replication through its interaction with viral Nef and matrix proteins, reduction of TNFα-induced signaling through its interaction with NF-κB pathway proteins, and corepression of agonist-bound retinoic acid receptors and peroxisome proliferator-activated receptors (PPAR). The wide tissue distribution of TNIP1 provides the opportunity to influence numerous cellular responses in these roles and defining control of TNIP1 expression would be central to improved understanding of its impact on cell function. We cloned 6kb of the human TNIP1 promoter and performed predictive and functional analyses to identify regulatory elements. The promoter region proximal to the transcription start site is GC-rich without a recognizable TATA box. In contrast to this proximal ~500bp region, 6kb of the promoter increased reporter construct constitutive activity over five-fold. Throughout the 6kb length, in silico analysis identified several potential binding sites for both constitutive and inducible transcription factors; among the latter were candidate NF-κB binding sequences and peroxisome proliferator response elements (PPREs). We tested NF-κB and PPAR regulation of the endogenous TNIP1 gene and cloned promoter by expression studies, electrophoretic mobility shift assays, and chromatin immunoprecipitations. We validated NF-κB sites in the TNIP1 promoter proximal and distal regions as well as one PPRE in the distal region. The ultimate control of the TNIP1 promoter is likely to be a combination of constitutive transcription factors and those subject to activation such as NF-κB and PPAR. PMID:22001530

Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads.

PubMed

Rombel, I T; McMorran, B J; Lamont, I L

1995-02-20

Many bacteria respond to a lack of iron in the environment by synthesizing siderophores, which act as iron-scavenging compounds. Fluorescent pseudomonads synthesize strain-specific but chemically related siderophores called pyoverdines or pseudobactins. We have investigated the mechanisms by which iron controls expression of genes involved in pyoverdine metabolism in Pseudomonas aeruginosa. Transcription of these genes is repressed by the presence of iron in the growth medium. Three promoters from these genes were cloned and the activities of the promoters were dependent on the amounts of iron in the growth media. Two of the promoters were sequenced and the transcriptional start site were identified by S1 nuclease analysis. Sequences similar to the consensus binding site for the Fur repressor protein, which controls expression of iron-repressible genes in several gram-negative species, were not present in the promoters, suggesting that they are unlikely to have a high affinity for Fur. However, comparison of the promoter sequences with those of iron-regulated genes from other Pseudomonas species and also the iron-regulated exotoxin gene of P. aeruginosa allowed identification of a shared sequence element, with the consensus sequence (G/C)CTAAAT-CCC, which is likely to act as a binding site for a transcriptional activator protein. Mutations in this sequence greatly reduced the activities of the promoters characterized here as well as those of other iron-regulated promoters. The requirement for this motif in the promoters of iron-regulated genes of different Pseudomonas species indicates that similar mechanisms are likely to be involved in controlling expression of a range of iron-regulated genes in pseudomonads.

Sort-Seq Approach to Engineering a Formaldehyde-Inducible Promoter for Dynamically Regulated Escherichia coli Growth on Methanol

PubMed Central

2017-01-01

Tight and tunable control of gene expression is a highly desirable goal in synthetic biology for constructing predictable gene circuits and achieving preferred phenotypes. Elucidating the sequence–function relationship of promoters is crucial for manipulating gene expression at the transcriptional level, particularly for inducible systems dependent on transcriptional regulators. Sort-seq methods employing fluorescence-activated cell sorting (FACS) and high-throughput sequencing allow for the quantitative analysis of sequence–function relationships in a robust and rapid way. Here we utilized a massively parallel sort-seq approach to analyze the formaldehyde-inducible Escherichia coli promoter (Pfrm) with single-nucleotide resolution. A library of mutated formaldehyde-inducible promoters was cloned upstream of gfp on a plasmid. The library was partitioned into bins via FACS on the basis of green fluorescent protein (GFP) expression level, and mutated promoters falling into each expression bin were identified with high-throughput sequencing. The resulting analysis identified two 19 base pair repressor binding sites, one upstream of the −35 RNA polymerase (RNAP) binding site and one overlapping with the −10 site, and assessed the relative importance of each position and base therein. Key mutations were identified for tuning expression levels and were used to engineer formaldehyde-inducible promoters with predictable activities. Engineered variants demonstrated up to 14-fold lower basal expression, 13-fold higher induced expression, and a 3.6-fold stronger response as indicated by relative dynamic range. Finally, an engineered formaldehyde-inducible promoter was employed to drive the expression of heterologous methanol assimilation genes and achieved increased biomass levels on methanol, a non-native substrate of E. coli. PMID:28463494

Structural insights into alternative splicing-mediated desensitization of jasmonate signaling.

PubMed

Zhang, Feng; Ke, Jiyuan; Zhang, Li; Chen, Rongzhi; Sugimoto, Koichi; Howe, Gregg A; Xu, H Eric; Zhou, Mingguo; He, Sheng Yang; Melcher, Karsten

2017-02-14

Jasmonate ZIM-domain (JAZ) transcriptional repressors play a key role in regulating jasmonate (JA) signaling in plants. Below a threshold concentration of jasmonoyl isoleucine (JA-Ile), the active form of JA, the C-terminal Jas motif of JAZ proteins binds MYC transcription factors to repress JA signaling. With increasing JA-Ile concentration, the Jas motif binds to JA-Ile and the COI1 subunit of the SCF COI1 E3 ligase, which mediates ubiquitination and proteasomal degradation of JAZ repressors, resulting in derepression of MYC transcription factors. JA signaling subsequently becomes desensitized, in part by feedback induction of JAZ splice variants that lack the C-terminal Jas motif but include an N-terminal cryptic MYC-interaction domain (CMID). The CMID sequence is dissimilar to the Jas motif and is incapable of recruiting SCF COI1 , allowing CMID-containing JAZ splice variants to accumulate in the presence of JA and to re-repress MYC transcription factors as an integral part of reestablishing signal homeostasis. The mechanism by which the CMID represses MYC transcription factors remains elusive. Here we describe the crystal structure of the MYC3-CMID JAZ10 complex. In contrast to the Jas motif, which forms a single continuous helix when bound to MYC3, the CMID adopts a loop-helix-loop-helix architecture with modular interactions with both the Jas-binding groove and the backside of the Jas-interaction domain of MYC3. This clamp-like interaction allows the CMID to bind MYC3 tightly and block access of MED25 (a subunit of the Mediator coactivator complex) to the MYC3 transcriptional activation domain, shedding light on the enigmatic mechanism by which JAZ splice variants desensitize JA signaling.

Streptomyces coelicolor encodes a urate-responsive transcriptional regulator with homology to PecS from plant pathogens.

PubMed

Huang, Hao; Mackel, Brian J; Grove, Anne

2013-11-01

Many transcriptional regulators control gene activity by responding to specific ligands. Members of the multiple-antibiotic resistance regulator (MarR) family of transcriptional regulators feature prominently in this regard, and they frequently function as repressors in the absence of their cognate ligands. Plant pathogens such as Dickeya dadantii encode a MarR homolog named PecS that controls expression of a gene encoding the efflux pump PecM in addition to other virulence genes. We report here that the soil bacterium Streptomyces coelicolor also encodes a PecS homolog (SCO2647) that regulates a pecM gene (SCO2646). S. coelicolor PecS, which exists as a homodimer, binds the intergenic region between pecS and pecM genes with high affinity. Several potential PecS binding sites were found in this intergenic region. The binding of PecS to its target DNA can be efficiently attenuated by the ligand urate, which also quenches the intrinsic fluorescence of PecS, indicating a direct interaction between urate and PecS. In vivo measurement of gene expression showed that activity of pecS and pecM genes is significantly elevated after exposure of S. coelicolor cultures to urate. These results indicate that S. coelicolor PecS responds to the ligand urate by attenuated DNA binding in vitro and upregulation of gene activity in vivo. Since production of urate is associated with generation of reactive oxygen species by xanthine dehydrogenase, we propose that PecS functions under conditions of oxidative stress.

Streptomyces coelicolor Encodes a Urate-Responsive Transcriptional Regulator with Homology to PecS from Plant Pathogens

PubMed Central

Huang, Hao; Mackel, Brian J.

2013-01-01

Many transcriptional regulators control gene activity by responding to specific ligands. Members of the multiple-antibiotic resistance regulator (MarR) family of transcriptional regulators feature prominently in this regard, and they frequently function as repressors in the absence of their cognate ligands. Plant pathogens such as Dickeya dadantii encode a MarR homolog named PecS that controls expression of a gene encoding the efflux pump PecM in addition to other virulence genes. We report here that the soil bacterium Streptomyces coelicolor also encodes a PecS homolog (SCO2647) that regulates a pecM gene (SCO2646). S. coelicolor PecS, which exists as a homodimer, binds the intergenic region between pecS and pecM genes with high affinity. Several potential PecS binding sites were found in this intergenic region. The binding of PecS to its target DNA can be efficiently attenuated by the ligand urate, which also quenches the intrinsic fluorescence of PecS, indicating a direct interaction between urate and PecS. In vivo measurement of gene expression showed that activity of pecS and pecM genes is significantly elevated after exposure of S. coelicolor cultures to urate. These results indicate that S. coelicolor PecS responds to the ligand urate by attenuated DNA binding in vitro and upregulation of gene activity in vivo. Since production of urate is associated with generation of reactive oxygen species by xanthine dehydrogenase, we propose that PecS functions under conditions of oxidative stress. PMID:23995633

DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor.

PubMed

Vonderach, Matthias; Byrne, Dominic P; Barran, Perdita E; Eyers, Patrick A; Eyers, Claire E

2018-06-05

The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKA c ) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKA c - and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKA c -regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κB DNA. Graphical Abstract ᅟ.

Biofilm Formation and Dispersal under the Influence of the Global Regulator CsrA of Escherichia coli

PubMed Central

Jackson, Debra W.; Suzuki, Kazushi; Oakford, Lawrence; Simecka, Jerry W.; Hart, Mark E.; Romeo, Tony

2002-01-01

The predominant mode of growth of bacteria in the environment is within sessile, matrix-enclosed communities known as biofilms. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system, decreasing antibiotic efficacy, and dispersing planktonic cells to distant body sites. While the biology of bacterial biofilms has become a major focus of microbial research, the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. Ectopic expression of the E. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. A csrA knockout mutation enhanced biofilm formation in E. coli strains that were defective for extracellular, surface, or regulatory factors previously implicated in biofilm formation. In contrast, this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. Complementation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Finally, the expression of a chromosomally encoded csrA′-′lacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E. coli biofilm development. PMID:11741870

Reassessment of the Genetic Regulation of Fatty Acid Synthesis in Escherichia coli: Global Positive Control by the Dual Functional Regulator FadR

PubMed Central

My, L.; Ghandour Achkar, N.; Viala, J. P.

2015-01-01

ABSTRACT In Escherichia coli, the FadR transcriptional regulator represses the expression of fatty acid degradation (fad) genes. However, FadR is also an activator of the expression of fabA and fabB, two genes involved in unsaturated fatty acid synthesis. Therefore, FadR plays an important role in maintaining the balance between saturated and unsaturated fatty acids in the membrane. We recently showed that FadR also activates the promoter upstream of the fabH gene (L. My, B. Rekoske, J. J. Lemke, J. P. Viala, R. L. Gourse, and E. Bouveret, J Bacteriol 195:3784–3795, 2013, doi:10.1128/JB.00384-13). Furthermore, recent transcriptomic and proteomic data suggested that FadR activates the majority of fatty acid (FA) synthesis genes. In the present study, we tested the role of FadR in the expression of all genes involved in FA synthesis. We found that FadR activates the transcription of all tested FA synthesis genes, and we identified the FadR binding site for each of these genes. This necessitated the reassessment of the transcription start sites for accA and accB genes described previously, and we provide evidence for the presence of multiple promoters driving the expression of these genes. We showed further that regulation by FadR impacts the amount of FA synthesis enzymes in the cell. Our results show that FadR is a global regulator of FA metabolism in E. coli, acting both as a repressor of catabolism and an activator of anabolism, two directly opposing pathways. IMPORTANCE In most bacteria, a transcriptional regulator tunes the level of FA synthesis enzymes. Oddly, such a global regulator still was missing for E. coli, which nonetheless is one of the prominent model bacteria used for engineering biofuel production using the FA synthesis pathway. Our work identifies the FadR functional dual regulator as a global activator of almost all FA synthesis genes in E. coli. Because FadR also is the repressor of FA degradation, FadR acts both as a repressor and an activator of the two opposite pathways of FA degradation and synthesis. Our results show that there are still discoveries waiting to be made in the understanding of the genetic regulation of FA synthesis, even in the very well-known bacterium E. coli. PMID:25802297

Expression of a repressor form of the Arabidopsis thaliana transcription factor TCP16 induces the formation of ectopic meristems.

PubMed

Uberti-Manassero, Nora G; Coscueta, Ezequiel R; Gonzalez, Daniel H

2016-11-01

Plants that express a fusion of the Arabidopsis thaliana class I TCP transcription factor TCP16 to the EAR repressor domain develop several phenotypic alterations, including rounder leaves, short petioles and pedicels, and delayed elongation of sepals, petals and anthers. In addition, these plants develop lobed cotyledons and ectopic meristems. Ectopic meristems are formed on the adaxial side of cotyledon petioles and arise from a cleft that is formed at this site. Analysis of the expression of reporter genes indicated that meristem genes are reactivated at the site of emergence of ectopic meristems, located near the bifurcation of cotyledon veins. The plants also show increased transcript levels of the boundary-specific CUP-SHAPED COTYLEDON (CUC) genes. The results suggest that TCP16 is able to modulate the induction of meristematic programs and the differentiation state of plant cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

A mutation-led search for novel functional domains in MeCP2.

PubMed

Guy, Jacky; Alexander-Howden, Beatrice; FitzPatrick, Laura; DeSousa, Dina; Koerner, Martha V; Selfridge, Jim; Bird, Adrian

2018-04-27

Most missense mutations causing Rett syndrome affect domains of MeCP2 that have been shown to either bind methylated DNA or interact with a transcriptional co-repressor complex. Several mutations, however, including the C-terminal truncations that account for ∼10% of cases, fall outside these characterised domains. We studied the molecular consequences of four of these "non-canonical" mutations in cultured neurons and mice to see if they reveal additional essential domains without affecting known properties of MeCP2. The results show that the mutations partially or strongly deplete the protein and also in some cases interfere with co-repressor recruitment. These mutations therefore impact the activity of known functional domains and do not invoke new molecular causes of Rett syndrome. The finding that a stable C-terminal truncation does not compromise MeCP2 function raises the possibility that small molecules which stabilise these mutant proteins may be of therapeutic value.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: an antibiotic target.

PubMed

Pendini, Nicole R; Yap, Min Y; Traore, D A K; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-06-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5'-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. © 2013 The Protein Society.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

PubMed Central

Pendini, Nicole R; Yap, Min Y; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-01-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present small-angle X-ray scattering data of SaBPL in complex with its biotin-carboxyl carrier protein substrate as well as the SaBPL:DNA complex that underlies repression. This has revealed the molecular basis of ligand (biotinyl-5′-AMP) binding and conformational changes associated with catalysis and repressor function. These data provide new information to better understand the bifunctional activities of SaBPL and to inform future strategies for antibiotic discovery. PMID:23559560

Application of TALE-Based Approach for Dissecting Functional MicroRNA-302/367 in Cellular Reprogramming.

PubMed

Zhang, Zhonghui; Wu, Wen-Shu

2018-01-01

MicroRNAs are small 18-24 nt single-stranded noncoding RNA molecules involved in many biological processes, including stemness maintenance and cellular reprogramming. Current methods used in loss-of-function studies of microRNAs have several limitations. Here, we describe a new approach for dissecting miR-302/367 functions by transcription activator-like effectors (TALEs), which are natural effector proteins secreted by Xanthomonas and Ralstonia bacteria. Knockdown of the miR-302/367 cluster uses the Kruppel-associated box repressor domain fused with specific TALEs designed to bind the miR-302/367 cluster promoter. Knockout of the miR-302/367 cluster uses two pairs of TALE nucleases (TALENs) to delete the miR-302/367 cluster in human primary cells. Together, both TALE-based transcriptional repressor and TALENs are two promising approaches for loss-of-function studies of microRNA cluster in human primary cells.

Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: Potential implications for methylation-independent transcriptional repression

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

Horton, John R.; Zhang, Xing; Blumenthal, Robert M.

DNA adenine methyltransferase (Dam) is widespread and conserved among the γ-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam and the promoter-proximal presence of GATC sequences. Here, we demonstrate that EcoDam interacts with a 5-base pair non-cognate sequence distinct from GATC. The crystal structure of a non-cognate complex allowed us to identify amore » DNA binding element, GTYTA/TARAC (where Y = C/T and R = A/G). This element immediately flanks GATC sites in some Dam-regulated promoters, including the Pap operon which specifies pyelonephritis-associated pili. In addition, Dam interacts with near-cognate GATC sequences (i.e. 3/4-site ATC and GAT). All together, these results imply that Dam, in addition to being responsible for GATC methylation, could also function as a methylation-independent transcriptional repressor.« less

Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: Potential implications for methylation-independent transcriptional repression

DOE PAGES

Horton, John R.; Zhang, Xing; Blumenthal, Robert M.; ...

2015-04-06

DNA adenine methyltransferase (Dam) is widespread and conserved among the γ-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam and the promoter-proximal presence of GATC sequences. Here, we demonstrate that EcoDam interacts with a 5-base pair non-cognate sequence distinct from GATC. The crystal structure of a non-cognate complex allowed us to identify amore » DNA binding element, GTYTA/TARAC (where Y = C/T and R = A/G). This element immediately flanks GATC sites in some Dam-regulated promoters, including the Pap operon which specifies pyelonephritis-associated pili. In addition, Dam interacts with near-cognate GATC sequences (i.e. 3/4-site ATC and GAT). All together, these results imply that Dam, in addition to being responsible for GATC methylation, could also function as a methylation-independent transcriptional repressor.« less

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

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. © 2016 Zentella et al.; Published by Cold Spring Harbor Laboratory Press.

A stationary-phase protein of Escherichia coli that affects the mode of association between the trp repressor protein and operator-bearing DNA.

PubMed

Yang, W; Ni, L; Somerville, R L

1993-06-15

Highly purified preparations of trp repressor (TrpR) protein derived from Escherichia coli strains that were engineered to overexpress this material were found to contain another protein, of 21 kDa. The second protein, designated WrbA [for tryptophan (W) repressor-binding protein] remained associated with its namesake through several sequential protein fractionation steps. The N-terminal amino acid sequence of the WrbA protein guided the design of two degenerate oligonucleotides that were used as probes in the cloning of the wrbA gene (198 codons). The WrbA protein, in purified form, was found by several criteria to enhance the formation and/or stability of noncovalent complexes between TrpR holorepressor and its primary operator targets. The formation of an operator-holorepressor-WrbA ternary complex was demonstrated by gel mobility-shift analysis. The WrbA protein alone does not interact with the trp operator. During the stationary phase, cells deficient in the WrbA protein were less efficient than wild type in their ability to repress the trp promoter. It is proposed that the WrbA protein functions as an accessory element in blocking TrpR-specific transcriptional processes that might be physiologically disadvantageous in the stationary phase of the bacterial life cycle.

Dual Roles for Ikaros in Regulation of Macrophage Chromatin State and Inflammatory Gene Expression.

PubMed

Oh, Kyu-Seon; Gottschalk, Rachel A; Lounsbury, Nicolas W; Sun, Jing; Dorrington, Michael G; Baek, Songjoon; Sun, Guangping; Wang, Ze; Krauss, Kathleen S; Milner, Joshua D; Dutta, Bhaskar; Hager, Gordon L; Sung, Myong-Hee; Fraser, Iain D C

2018-06-13

Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor families. The transcription factor Ikaros has been shown to play a critical role in lymphoid cell development and differentiation; however, its function in myeloid cells and innate immune responses is less appreciated. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1 -/- macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes, and chromatin immunoprecipitation sequencing analyses support a key role for Ikaros in sustained NF-κB chromatin binding. Decreased Ikaros expression in Ikzf1 +/- mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.

A MYB/ZML Complex Regulates Wound-Induced Lignin Genes in Maize

PubMed Central

Vélez-Bermúdez, Isabel-Cristina; Salazar-Henao, Jorge E.; Franco-Zorrilla, José-Manuel; Grotewold, Erich; Solano, Roberto

2015-01-01

Lignin is an essential polymer in vascular plants that plays key structural roles in vessels and fibers. Lignification is induced by external inputs such as wounding, but the molecular mechanisms that link this stress to lignification remain largely unknown. In this work, we provide evidence that three maize (Zea mays) lignin repressors, MYB11, MYB31, and MYB42, participate in wound-induced lignification by interacting with ZML2, a protein belonging to the TIFY family. We determined that the three R2R3-MYB factors and ZML2 bind in vivo to AC-rich and GAT(A/C) cis-elements, respectively, present in a set of lignin genes. In particular, we show that MYB11 and ZML2 bind simultaneously to the AC-rich and GAT(A/C) cis-elements present in the promoter of the caffeic acid O-methyl transferase (comt) gene. We show that, like the R2R3-MYB factors, ZML2 also acts as a transcriptional repressor. We found that upon wounding and methyl jasmonate treatments, MYB11 and ZML2 proteins are degraded and comt transcription is induced. Based on these results, we propose a molecular regulatory mechanism involving a MYB/ZML complex in which wound-induced lignification can be achieved by the derepression of a set of lignin genes. PMID:26566917

The WRKY57 Transcription Factor Affects the Expression of Jasmonate ZIM-Domain Genes Transcriptionally to Compromise Botrytis cinerea Resistance.

PubMed

Jiang, Yanjuan; Yu, Diqiu

2016-08-01

Although necrotrophic pathogens cause many devastating plant diseases, our understanding of the plant defense response to them is limited. Here, we found that loss of function of WRKY57 enhanced the resistance of Arabidopsis (Arabidopsis thaliana) against Botrytis cinerea infection. Further investigation suggested that the negative regulation of WRKY57 against B cinerea depends on the jasmonic acid (JA) signaling pathway. Chromatin immunoprecipitation experiments revealed that WRKY57 directly binds to the promoters of JASMONATE ZIM-DOMAIN1 (JAZ1) and JAZ5, encoding two important repressors of the JA signaling pathway, and activates their transcription. In vivo and in vitro experiments demonstrated that WRKY57 interacts with nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2. Further experiments display that the same domain, the VQ motif, of SIB1 and SIB2 interact with WRKY33 and WRKY57. Moreover, transient transcriptional activity assays confirmed that WRKY57 and WRKY33 competitively regulate JAZ1 and JAZ5, SIB1 and SIB2 further enhance these competitions of WRKY57 to WRKY33. Therefore, coordinated regulation of Arabidopsis against B cinerea by transcription activators and repressors would benefit plants by allowing fine regulation of defense. © 2016 American Society of Plant Biologists. All Rights Reserved.

Quaternary re-arrangement analysed by spectral enhancement: the interaction of a sporulation repressor with its antagonist.

PubMed

Scott, D J; Leejeerajumnean, S; Brannigan, J A; Lewis, R J; Wilkinson, A J; Hoggett, J G

1999-11-12

The protein/protein interaction between SinI and SinR has been studied by analytical ultracentrifugation and gel electrophoresis in an attempt to understand how these proteins contribute to developmental control of sporulation in Bacillus subtilis. SinR was found to be tetrameric, while SinI was found to exist as monomers and dimers in a rapidly reversible equilibrium. Labelling of SinR by incorporating the tryptophan analogue 7-azatryptophan (7AW) into the protein in place of tryptophan shifts the UV absorbance spectrum, thus allowing selective monitoring of 7AWSinR at 315 nm using the UV absorption optics of the analytical ultracentrifuge. Selective monitoring of SinR in mixtures of SinR and SinI enables the binding and stoichiometry of the interaction to be investigated quantitatively and unambiguously. We demonstrate that the oligomeric forms of SinR and SinI re-arrange to form a tight 1:1 SinR:SinI complex, with no stable intermediate species. A fragment of SinR, SinR(1-69), which contains only the DNA-binding domain, was found to be monomeric, showing that the protein appears not to oligomerise in a similar manner to the Cro repressor, a protein with which it shares a marked structural similarity. Copyright 1999 Academic Press.

The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

PubMed

Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela

2017-09-06

In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.

Bats: Body mass index, forearm mass index, blood glucose levels and SLC2A2 genes for diabetes

PubMed Central

Meng, Fanxing; Zhu, Lei; Huang, Wenjie; Irwin, David M.; Zhang, Shuyi

2016-01-01

Bats have an unusually large volume of endocrine tissue, with a large population of beta cells, and an elevated sensitivity to glucose and insulin. This makes them excellent animal models for studying diabetes mellitus. We evaluated bats as models for diabetes in terms of lifestyle and genetic factors. For lifestyle factors, we generated data sets of 149 body mass index (BMI) and 860 forearm mass index (FMI) measurements for different species of bats. Both showed negative inter-species correlations with blood glucose levels in sixteen bats examined. The negative inter-species correlations may reflect adaptation of a small insectivorous ancestor to a larger frugivore. We identified an 11 bp deletion in the proximal promoter of SLC2A2 that we predicted would disrupt binding sites for the transcription repressor ZNF354C. In frugivorous bats this could explain the relatively high expression of this gene, resulting in a better capacity to absorb glucose and decrease blood glucose levels. PMID:27439361

AML1/ETO accelerates cell migration and impairs cell-to-cell adhesion and homing of hematopoietic stem/progenitor cells

PubMed Central

Saia, Marco; Termanini, Alberto; Rizzi, Nicoletta; Mazza, Massimiliano; Barbieri, Elisa; Valli, Debora; Ciana, Paolo; Gruszka, Alicja M.; Alcalay, Myriam

2016-01-01

The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression. PMID:27713544

A novel herbicide-inducible male sterility system.

PubMed

Zhang, Jinhui; Zhang, Wenlu; Yen, Yang; Long, Hai; Deng, Guangbing; Pan, Zhifen; Yu, Maoqun

2010-11-01

Heterosis is a phenomenon that first-generation offspring perform better than their parents. Conventional breeding methods have their shortcomings. It would be optimal to construct inducible male sterile plants. We developed a novel system for creating male sterile transgenic plants by downregulating the specific expression of the glyphosate tolerance CP4 EPSPS gene in male reproductive tissues. Transcriptional repression was achieved by manipulating DNA binding proteins with their specific corresponding sites. We transferred the CP4 EPSPS gene driven by a modified CaMV 35S promoter with three tetracycline operator copies in the vicinity of the TATA box and tetracycline repressor gene under the control of an anther-specific promoter Osg6B to Arabidopsis thaliana. As a result, we successfully obtained controllable transgenic plants: the whole plant could tolerate exposure of glyphosate but the male tissue was sensitive. The novel inducible male sterility system is applied and easy to handle, so it might be applicable to a wide range of crop plants. 2010 Society of Chemical Industry

Exploring the sequence-function relationship in transcriptional regulation by the lac O1 operator.

PubMed

Maity, Tuhin S; Jha, Ramesh K; Strauss, Charlie E M; Dunbar, John

2012-07-01

Understanding how binding of a transcription factor to an operator is influenced by the operator sequence is an ongoing quest. It facilitates discovery of alternative binding sites as well as tuning of transcriptional regulation. We investigated the behavior of the Escherichia coli Lac repressor (LacI) protein with a large set of lac O(1) operator variants. The 114 variants examined contained a mean of 2.9 (range 0-4) mutations at positions -4, -2, +2 and +4 in the minimally required 17 bp operator. The relative affinity of LacI for the operators was examined by quantifying expression of a GFP reporter gene and Rosetta structural modeling. The combinations of mutations in the operator sequence created a wide range of regulatory behaviors. We observed variations in the GFP fluorescent signal among the operator variants of more than an order of magnitude under both uninduced and induced conditions. We found that a single nucleotide change may result in changes of up to six- and 12-fold in uninduced and induced GFP signals, respectively. Among the four positions mutated, we found that nucleotide G at position -4 is strongly correlated with strong repression. By Rosetta modeling, we found a significant correlation between the calculated binding energy and the experimentally observed transcriptional repression strength for many operators. However, exceptions were also observed, underscoring the necessity for further improvement in biophysical models of protein-DNA interactions. © 2012 The Authors Journal compilation © 2012 FEBS.

Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

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

Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha

2014-07-18

Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated thatmore » TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations.« less

The Yeast PUF Protein Puf5 Has Pop2-Independent Roles in Response to DNA Replication Stress

PubMed Central

Traven, Ana; Lo, Tricia L.; Lithgow, Trevor; Heierhorst, Jörg

2010-01-01

PUFs are RNA binding proteins that promote mRNA deadenylation and decay and inhibit translation. Yeast Puf5 is the prototype for studying PUF-dependent gene repression. Puf5 binds to the Pop2 subunit of the Ccr4-Pop2-NOT mRNA deadenylase, recruiting the deadenylase and associated translational repressors to mRNAs. Here we used yeast genetics to show that Puf5 has additional roles in vivo that do not require Pop2. Deletion of PUF5 caused increased sensitivity to DNA replication stress in cells lacking Pop2, as well as in cells mutated for two activities recruited to mRNAs by the Puf5-Pop2 interaction, the deadenylase Ccr4 and the translational repressor Dhh1. A functional Puf5 RNA binding domain was required, and Puf5 cytoplasmic localisation was sufficient for resistance to replication stress, indicating posttranscriptional gene expression control is involved. In contrast to DNA replication stress, in response to the cell wall integrity pathway activator caffeine, PUF5 and POP2 acted in the same genetic pathway, indicating that functions of Puf5 in the caffeine response are mediated by Pop2-dependent gene repression. Our results support a model in which Puf5 uses multiple, Pop2-dependent and Pop2-independent mechanisms to control mRNA expression. The Pop2-independent roles for Puf5 could involve spatial control of gene expression, a proposition supported by our data indicating that the active form of Puf5 is localised to cytoplasmic foci. PMID:20498834

Opi1 mediates repression of phospholipid biosynthesis by phosphate limitation in the yeast Saccharomyces cerevisiae.

PubMed

Kliewe, Felix; Kumme, Jacqueline; Grigat, Mathias; Hintze, Stefan; Schüller, Hans-Joachim

2017-02-01

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are transcribed when precursor molecules inositol and choline (IC) are limiting. Gene expression is stimulated by the heterodimeric activator Ino2/Ino4, which binds to ICRE (inositol/choline-responsive element) promoter sequences. Activation is prevented by repressor Opi1, counteracting Ino2 when high concentrations of IC are available. Here we show that ICRE-dependent gene activation is repressed not only by an excess of IC but also under conditions of phosphate starvation. While PHO5 is activated by phosphate limitation, INO1 expression is repressed about 10-fold. Repression of ICRE-dependent genes by low phosphate is no longer observed in an opi1 mutant while repression is still effective in mutants of the PHO regulon (pho4, pho80, pho81 and pho85). In contrast, gene expression with high phosphate is reduced in the absence of pleiotropic sensor protein kinase Pho85. We could demonstrate that Pho85 binds to Opi1 in vitro and in vivo and that this interaction is increased in the presence of high concentrations of phosphate. Interestingly, Pho85 binds to two separate domains of Opi1 which have been previously shown to recruit pleiotropic corepressor Sin3 and activator Ino2, respectively. We postulate that Pho85 positively influences ICRE-dependent gene expression by phosphorylation-dependent weakening of Opi1 repressor, affecting its functional domains required for promoter recruitment and corepressor interaction. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

A plausibly causal functional lupus-associated risk variant in the STAT1-STAT4 locus.

PubMed

Patel, Zubin; Lu, Xiaoming; Miller, Daniel; Forney, Carmy R; Lee, Joshua; Lynch, Arthur; Schroeder, Connor; Parks, Lois; Magnusen, Albert F; Chen, Xiaoting; Pujato, Mario; Maddox, Avery; Zoller, Erin E; Namjou, Bahram; Brunner, Hermine I; Henrickson, Michael; Huggins, Jennifer L; Williams, Adrienne H; Ziegler, Julie T; Comeau, Mary E; Marion, Miranda C; Glenn, Stuart B; Adler, Adam; Shen, Nan; Nath, Swapan K; Stevens, Anne M; Freedman, Barry I; Pons-Estel, Bernardo A; Tsao, Betty P; Jacob, Chaim O; Kamen, Diane L; Brown, Elizabeth E; Gilkeson, Gary S; Alarcón, Graciela S; Martin, Javier; Reveille, John D; Anaya, Juan-Manuel; James, Judith A; Sivils, Kathy L; Criswell, Lindsey A; Vilá, Luis M; Petri, Michelle; Scofield, R Hal; Kimberly, Robert P; Edberg, Jeffrey C; Ramsey-Goldman, Rosalind; Bang, So-Young; Lee, Hye-Soon; Bae, Sang-Cheol; Boackle, Susan A; Cunninghame Graham, Deborah; Vyse, Timothy J; Merrill, Joan T; Niewold, Timothy B; Ainsworth, Hannah C; Silverman, Earl D; Weisman, Michael H; Wallace, Daniel J; Raj, Prithvi; Guthridge, Joel M; Gaffney, Patrick M; Kelly, Jennifer A; Alarcón-Riquelme, Marta E; Langefeld, Carl D; Wakeland, Edward K; Kaufman, Kenneth M; Weirauch, Matthew T; Harley, John B; Kottyan, Leah C

2018-04-18

Systemic Lupus Erythematosus (SLE or lupus) (OMIM: 152700) is a chronic autoimmune disease with debilitating inflammation that affects multiple organ systems. The STAT1-STAT4 locus is one of the first and most highly-replicated genetic loci associated with lupus risk. We performed a fine-mapping study to identify plausible causal variants within the STAT1-STAT4 locus associated with increased lupus disease risk. Using complementary frequentist and Bayesian approaches in trans-ancestral Discovery and Replication cohorts, we found one variant whose association with lupus risk is supported across ancestries in both the Discovery and Replication cohorts: rs11889341. In B cell lines from patients with lupus and healthy controls, the lupus risk allele of rs11889341 was associated with increased STAT1 expression. We demonstrated that the transcription factor HMGA1, a member of the HMG transcription factor family with an AT-hook DNA-binding domain, has enriched binding to the risk allele compared to the non-risk allele of rs11889341. We identified a genotype-dependent repressive element in the DNA within the intron of STAT4 surrounding rs11889341. Consistent with expression quantitative trait locus (eQTL) analysis, the lupus risk allele of rs11889341 decreased the activity of this putative repressor. Altogether, we present a plausible molecular mechanism for increased lupus risk at the STAT1-STAT4 locus in which the risk allele of rs11889341, the most probable causal variant, leads to elevated STAT1 expression in B cells due to decreased repressor activity mediated by increased binding of HMGA1.

Sequestration of cAMP response element-binding proteins by transcription factor decoys causes collateral elaboration of regenerating Aplysia motor neuron axons.

PubMed

Dash, P K; Tian, L M; Moore, A N

1998-07-07

Axonal injury increases intracellular Ca2+ and cAMP and has been shown to induce gene expression, which is thought to be a key event for regeneration. Increases in intracellular Ca2+ and/or cAMP can alter gene expression via activation of a family of transcription factors that bind to and modulate the expression of CRE (Ca2+/cAMP response element) sequence-containing genes. We have used Aplysia motor neurons to examine the role of CRE-binding proteins in axonal regeneration after injury. We report that axonal injury increases the binding of proteins to a CRE sequence-containing probe. In addition, Western blot analysis revealed that the level of ApCREB2, a CRE sequence-binding repressor, was enhanced as a result of axonal injury. The sequestration of CRE-binding proteins by microinjection of CRE sequence-containing plasmids enhanced axon collateral formation (both number and length) as compared with control plasmid injections. These findings show that Ca2+/cAMP-mediated gene expression via CRE-binding transcription factors participates in the regeneration of motor neuron axons.

An incoherent feedforward loop facilitates adaptive tuning of gene expression.

PubMed

Hong, Jungeui; Brandt, Nathan; Abdul-Rahman, Farah; Yang, Ally; Hughes, Tim; Gresham, David

2018-04-05

We studied adaptive evolution of gene expression using long-term experimental evolution of Saccharomyces cerevisiae in ammonium-limited chemostats. We found repeated selection for non-synonymous variation in the DNA binding domain of the transcriptional activator, GAT1, which functions with the repressor, DAL80 in an incoherent type-1 feedforward loop (I1-FFL) to control expression of the high affinity ammonium transporter gene, MEP2. Missense mutations in the DNA binding domain of GAT1 reduce its binding to the GATAA consensus sequence. However, we show experimentally, and using mathematical modeling, that decreases in GAT1 binding result in increased expression of MEP2 as a consequence of properties of I1-FFLs. Our results show that I1-FFLs, one of the most commonly occurring network motifs in transcriptional networks, can facilitate adaptive tuning of gene expression through modulation of transcription factor binding affinities. Our findings highlight the importance of gene regulatory architectures in the evolution of gene expression. © 2018, Hong et al.

Building a dictionary for genomes: Identification of presumptive regulatory sites by statistical analysis

PubMed Central

Bussemaker, Harmen J.; Li, Hao; Siggia, Eric D.

2000-01-01

The availability of complete genome sequences and mRNA expression data for all genes creates new opportunities and challenges for identifying DNA sequence motifs that control gene expression. An algorithm, “MobyDick,” is presented that decomposes a set of DNA sequences into the most probable dictionary of motifs or words. This method is applicable to any set of DNA sequences: for example, all upstream regions in a genome or all genes expressed under certain conditions. Identification of words is based on a probabilistic segmentation model in which the significance of longer words is deduced from the frequency of shorter ones of various lengths, eliminating the need for a separate set of reference data to define probabilities. We have built a dictionary with 1,200 words for the 6,000 upstream regulatory regions in the yeast genome; the 500 most significant words (some with as few as 10 copies in all of the upstream regions) match 114 of 443 experimentally determined sites (a significance level of 18 standard deviations). When analyzing all of the genes up-regulated during sporulation as a group, we find many motifs in addition to the few previously identified by analyzing the subclusters individually to the expression subclusters. Applying MobyDick to the genes derepressed when the general repressor Tup1 is deleted, we find known as well as putative binding sites for its regulatory partners. PMID:10944202

Metabolic Control of Virulence Genes in Brucella abortus: HutC Coordinates virB Expression and the Histidine Utilization Pathway by Direct Binding to Both Promoters ▿ †

PubMed Central

Sieira, Rodrigo; Arocena, Gastón M.; Bukata, Lucas; Comerci, Diego J.; Ugalde, Rodolfo A.

2010-01-01

Type IV secretion systems (T4SS) are multicomponent machineries involved in the translocation of effector molecules across the bacterial cell envelope. The virB operon of Brucella abortus codes for a T4SS that is essential for virulence and intracellular multiplication of the bacterium in the host. Previous studies showed that the virB operon of B. abortus is tightly regulated within the host cells. In order to identify factors implicated in the control of virB expression, we searched for proteins of Brucella that directly bind to the virB promoter (PvirB). Using different procedures, we isolated a 27-kDa protein that binds specifically to PvirB. This protein was identified as HutC, the transcriptional repressor of the histidine utilization (hut) genes. Analyses of virB and hut promoter activity revealed that HutC exerts two different roles: it acts as a coactivator of transcription of the virB operon, whereas it represses the hut genes. Such activities were observed both intracellularly and in bacteria incubated under conditions that resemble the intracellular environment. Electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments revealed the structure, affinity, and localization of the HutC-binding sites and supported the regulatory role of HutC in both hut and virB promoters. Taken together, these results indicate that Brucella coopted the function of HutC to coordinate the Hut pathway with transcriptional regulation of the virB genes, probably as a way to sense its own metabolic state and develop adaptive responses to overcome intracellular host defenses. PMID:19854911

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

PubMed Central

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

1994-01-01

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

The MicroRNA390/TRANS-ACTING SHORT INTERFERING RNA3 Module Mediates Lateral Root Growth under Salt Stress via the Auxin Pathway.

PubMed

He, Fu; Xu, Changzheng; Fu, Xiaokang; Shen, Yun; Guo, Li; Leng, Mi; Luo, Keming

2018-06-01

Salt-induced developmental plasticity in a plant root system strongly depends on auxin signaling. However, the molecular events underlying this process are poorly understood. MicroRNA390 ( miR390 ), trans-actin small interfering RNA s ( tasiRNA s), and AUXIN RESPONSE FACTORs ( ARFs ) form a regulatory module involved in controlling lateral root (LR) growth. Here, we found that miR390 expression was strongly induced by exposure to salt during LR formation in poplar ( Populus spp.) plants. miR390 overexpression stimulated LR development and increased salt tolerance, whereas miR390 knockdown caused by a short tandem target mimic repressed LR growth and compromised salt resistance. ARF3.1 , ARF3.2 , and ARF4 expression was inhibited significantly by the presence of salt, and transcript abundance was decreased dramatically in the miR390 -overexpressing line but increased in the miR390 -knockdown line. Constitutive expression of ARF4m harboring mutated trans-acting small interfering ARF -binding sites removed the salt resistance of the miR390 overexpressors. miR390 positively regulated auxin signaling in LRs subjected to salt, but ARF4 inhibited auxin signaling. Salinity stabilized the poplar Aux/IAA repressor INDOLE-3-ACETIC ACID17.1, and overexpression of an auxin/salt-resistant form of this repressor suppressed LR growth in miR390 -overexpressing and ARF4 -RNA interfering lines in the presence of salt. Thus, the miR390/TAS3/ARFs module is a key regulator, via modulating the auxin pathway, of LR growth in poplar subjected to salt stress. © 2018 American Society of Plant Biologists. All rights reserved.

microRNA 21-mediated suppression of Sprouty1 by Pokemon affects liver cancer cell growth and proliferation.

PubMed

Jin, Xiu-Li; Sun, Qin-Sheng; Liu, Feng; Yang, Hong-Wei; Liu, Min; Liu, Hong-Xia; Xu, Wei; Jiang, Yu-Yang

2013-07-01

Transcriptional repressor Pokemon is a critical factor in embryogenesis, development, cell proliferation, differentiation, and oncogenesis, thus behaving as an oncogene. Oncomine database suggests a potential correlation between the expressions of Pokemon and Sprouty1. This study investigated the regulatory role of Pokemon in Sprouty1 expression and the effect on liver cancer cell growth and proliferation, revealing a novel miR-21-mediated regulatory circuit. In normal (HL-7702) and cancer (QGY-7703) liver cell lines, Sprouty1 expression is inversely correlated with Pokemon levels. Targeted expression or siRNA-mediated silencing showed that Pokemon is a repressor of Sprouty1 expression at both mRNA and protein levels, but Pokemon cannot affect the promoter activity of Sprouty1. Sprouty1 is a target of miR-21 and interestingly, we found that miR-21 is up-regulated by Pokemon in liver cancer cells. Luciferase reporter assays showed that Pokemon up-regulated miR-21 transcription in a dose-dependent manner, and ChIP assay exhibited a direct binding of Pokemon to the miR-21 promoter at -747 to -399 bp. Site-directed mutagenesis of the GC boxes at -684 to -679 bp and -652 to -647 bp of miR-21 promoter abolished the regulatory activity by Pokemon. Furthermore, we found that the modulation of Pokemon and miR-21 expression affected the growth and proliferation of liver cancer cells QGY-7703. In summary, our findings demonstrate that Pokemon suppresses Sprouty1 expression through a miR-21-mediated mechanism, affecting the growth and proliferation of liver cancer cells. This study recognized miR-21 and Sprouty1 as novel targets of the Pokemon regulatory network. Copyright © 2013 Wiley Periodicals, Inc.

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

Krishnamurthy, Malathy; Hennelly, Scott Patrick; Dale, Taraka T.

The most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme catalyst. Until recently engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or by using one of several strategies to knock down or knock out a wasteful gene. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. We report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. Ourmore » design includes a cis-repressor at the 5’ end of the mRNA that forms a stem-loop helix occluding the ribosome binding site and blocking translation. An activating-RNA, expressed in trans, frees the RBS turning on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability followed by directed evolution on a portion of each trans-activator to fine tune translation. We report a cis-repressor that can completely shut off translation of antibiotic resistance reporters and a trans-activator that restores translation. We have shown it is possible to use riboregulators to achieve translational control of gene expression over a wide dynamic range. Using a bioluminescent reporter system, we demonstrated an ON/OFF ratio >300. We have demonstrated that a targeting sequence can be changed to develop riboregulators that can independently regulate translation of many genes with minimal cross-talk. In a SELEX experiment, we demonstrated that by subtly altering the sequence of the trans-activator, it is possible to alter the equilibrium between repressed and activated states and achieve intermediate translational control.« less

Tunable riboregulator switches for post-transcriptional control of gene expression

DOE PAGES

Krishnamurthy, Malathy; Hennelly, Scott Patrick; Dale, Taraka T.; ...

2015-07-13

The most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme catalyst. Until recently engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or by using one of several strategies to knock down or knock out a wasteful gene. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. We report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. Ourmore » design includes a cis-repressor at the 5’ end of the mRNA that forms a stem-loop helix occluding the ribosome binding site and blocking translation. An activating-RNA, expressed in trans, frees the RBS turning on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability followed by directed evolution on a portion of each trans-activator to fine tune translation. We report a cis-repressor that can completely shut off translation of antibiotic resistance reporters and a trans-activator that restores translation. We have shown it is possible to use riboregulators to achieve translational control of gene expression over a wide dynamic range. Using a bioluminescent reporter system, we demonstrated an ON/OFF ratio >300. We have demonstrated that a targeting sequence can be changed to develop riboregulators that can independently regulate translation of many genes with minimal cross-talk. In a SELEX experiment, we demonstrated that by subtly altering the sequence of the trans-activator, it is possible to alter the equilibrium between repressed and activated states and achieve intermediate translational control.« less

tif-dependent induction of colicin E1, prophage lambda, and filamentation in Escherichia coli K-12.

PubMed

Tessman, E S; Peterson, P K

1980-09-01

To help understand how the tif-1 mutation of the recA gene of Escherichia coli confers adenine activability on the recA protein, we used the fact that cytidine plus guanosine inhibits induction of prophage lambda and cell filamentation in a tif-1 mutant, and that adenine reverses this inhibition. We varied the amount of adenine in agar plates containing a fixed amount of cytidine and scored for survivors of three different tif-dependent lethal induction processes. Much more adenine was required for cell killing when cytidine was present than when it was absent. Therefore adenine does not override cytidine inhibition, but instead appears to compete with it for a site of action which may be on the recA protein. The competition is not at the cell transport level. Our results lead to a model in which the tif form of the recA protein is an allosteric enzyme that binds both negative and positive modulators. By varying the adenine-cytidine ratio of the medium it is possible to control the degree of induction in a tif-1 cell. For the three different tif-dependent inductions studied here, least adenine was required for lambda induction and most for lethal filamentation, presumably reflecting requirements for different amounts of activated recA protein in each process. Varying the adenine-cytidine ratio revealed two stable intermediate stages in lambda induction, as well as a stage of colicin E1 induction in which the cells produced colicin without cell death. The rate of filament formation could be similarly controlled. Experiments with tif (ColE1, lambda) gave evidence of a competition between colicin repressor and lambda repressor for activated recA protein.

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. © 2014 John Wiley & Sons Ltd.

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

PubMed Central

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

2014-01-01

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 crosslinks, including di-, tri- and tetrasulfide bonds, which allosterically inhibit cst operator DNA binding by CstR. PMID:25318663

A stationary-phase protein of Escherichia coli that affects the mode of association between the trp repressor protein and operator-bearing DNA.

PubMed Central

Yang, W; Ni, L; Somerville, R L

1993-01-01

Highly purified preparations of trp repressor (TrpR) protein derived from Escherichia coli strains that were engineered to overexpress this material were found to contain another protein, of 21 kDa. The second protein, designated WrbA [for tryptophan (W) repressor-binding protein] remained associated with its namesake through several sequential protein fractionation steps. The N-terminal amino acid sequence of the WrbA protein guided the design of two degenerate oligonucleotides that were used as probes in the cloning of the wrbA gene (198 codons). The WrbA protein, in purified form, was found by several criteria to enhance the formation and/or stability of noncovalent complexes between TrpR holorepressor and its primary operator targets. The formation of an operator-holorepressor-WrbA ternary complex was demonstrated by gel mobility-shift analysis. The WrbA protein alone does not interact with the trp operator. During the stationary phase, cells deficient in the WrbA protein were less efficient than wild type in their ability to repress the trp promoter. It is proposed that the WrbA protein functions as an accessory element in blocking TrpR-specific transcriptional processes that might be physiologically disadvantageous in the stationary phase of the bacterial life cycle. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:8516330

Isolating intrinsic noise sources in a stochastic genetic switch.

PubMed

Newby, Jay M

2012-01-01

The stochastic mutual repressor model is analysed using perturbation methods. This simple model of a gene circuit consists of two genes and three promotor states. Either of the two protein products can dimerize, forming a repressor molecule that binds to the promotor of the other gene. When the repressor is bound to a promotor, the corresponding gene is not transcribed and no protein is produced. Either one of the promotors can be repressed at any given time or both can be unrepressed, leaving three possible promotor states. This model is analysed in its bistable regime in which the deterministic limit exhibits two stable fixed points and an unstable saddle, and the case of small noise is considered. On small timescales, the stochastic process fluctuates near one of the stable fixed points, and on large timescales, a metastable transition can occur, where fluctuations drive the system past the unstable saddle to the other stable fixed point. To explore how different intrinsic noise sources affect these transitions, fluctuations in protein production and degradation are eliminated, leaving fluctuations in the promotor state as the only source of noise in the system. The process without protein noise is then compared to the process with weak protein noise using perturbation methods and Monte Carlo simulations. It is found that some significant differences in the random process emerge when the intrinsic noise source is removed.

A homozygous mutation in HESX1 is associated with evolving hypopituitarism due to impaired repressor-corepressor interaction

PubMed Central

Carvalho, Luciani R.; Woods, Kathryn S.; Mendonca, Berenice B.; Marcal, Nathalie; Zamparini, Andrea L.; Stifani, Stefano; Brickman, Joshua M.; Arnhold, Ivo J.P.; Dattani, Mehul T.

2003-01-01

The paired-like homeobox gene expressed in embryonic stem cells Hesx1/HESX1 encodes a developmental repressor and is expressed in early development in a region fated to form the forebrain, with subsequent localization to Rathke’s pouch, the primordium of the anterior pituitary gland. Mutations within the gene have been associated with septo-optic dysplasia, a constellation of phenotypes including eye, forebrain, and pituitary abnormalities, or milder degrees of hypopituitarism. We identified a novel homozygous nonconservative missense mutation (I26T) in the critical Engrailed homology repressor domain (eh1) of HESX1, the first, to our knowledge, to be described in humans, in a girl with evolving combined pituitary hormone deficiency born to consanguineous parents. Neuroimaging revealed a thin pituitary stalk with anterior pituitary hypoplasia and an ectopic posterior pituitary, but no midline or optic nerve abnormalities. This I26T mutation did not affect the DNA-binding ability of HESX1 but led to an impaired ability to recruit the mammalian Groucho homolog/Transducin-like enhancer of split-1 (Gro/TLE1), a crucial corepressor for HESX1, thereby leading to partial loss of repression. Thus, the novel pituitary phenotype highlighted here appears to be a specific consequence of the inability of HESX1 to recruit Groucho-related corepressors, suggesting that other molecular mechanisms govern HESX1 function in the forebrain. PMID:14561704

Repressive coping and self-reports of parenting.

PubMed

Myers, L B; Brewin, C R; Winter, D A

1999-03-01

To investigate whether women who possess a repressive coping style (repressors) self-report more positive judgments of their childhood on questionnaire and repertory grid measures compared with non-repressors. Repressors (low anxiety-high defensiveness) were compared with a composite group of non-repressors, containing some low anxious (low anxiety-low defensiveness), some high anxious (high anxiety-low defensiveness), some defensive high anxious (high anxiety-high defensiveness) and some non-extreme scorers. Participants completed the Parental Bonding Instrument (PBI; Parker, Tupling & Brown, 1979) and a 10 x 10 repertory grid, Self-Identification Form. On the PBI, repressors scored significantly higher than non-repressors on paternal care and significantly lower on paternal overprotection. There were no group differences for maternal measures. On the repertory grid, repressors compared with non-repressors perceived (a) themselves as significantly closer to their father, a woman they like, and their ideal partner, and significantly further from a woman they dislike, and a man they dislike; and (b) their father as significantly closer to a woman they like, a partner/person they admire, and an ideal partner. In addition, repressors were significantly tighter on construing than non-repressors. The results supported the hypothesis that repressors would rate their interactions with their fathers more positively than non-repressors when allowed to do so on self-report measures.

Trichostatin A Selectively Suppresses the Cold-Induced Transcription of the ZmDREB1 Gene in Maize

PubMed Central

Hu, Yong; Zhang, Lu; Zhao, Lin; Li, Jun; He, Shibin; Zhou, Kun; Yang, Fei; Huang, Min; Jiang, Li; Li, Lijia

2011-01-01

Post-translational modifications of histone proteins play a crucial role in responding to environmental stresses. Histone deacetylases (HDACs) catalyze the removal of an acetyl group from histones and are generally believed to be a transcriptional repressor. In this paper, we report that cold treatment highly induces the up-regulation of HDACs, leading to global deacetylation of histones H3 and H4. Treatment of maize with the HDAC inhibitor trichostatin A (TSA) under cold stress conditions strongly inhibits induction of the maize cold-responsive genes ZmDREB1 and ZmCOR413. However, up-regulation of the ZmICE1 gene in response to cold stress is less affected. The expression of drought and salt induced genes, ZmDBF1 and rab17, is almost unaffected by TSA treatment. Thus, these observations show that HDACs may selectively activate transcription. The time course of TSA effects on the expression of ZmDREB1 and ZmCOR413 genes indicates that HDACs appear to directly activate the ZmDREB1 gene, which in turn modulates ZmCOR413 expression. After cold treatment, histone hyperacetylation and DNA demethylation occurs in the ICE1 binding region, accompanied by an increase in accessibility to micrococcal nuclease (MNase). The two regions adjacent to the ICE1 binding site remain hypoacetylated and methylated. However, during cold acclimation, TSA treatment increases the acetylation status and accessibility of MNase and decreases DNA methylation at these two regions. However, TSA treatment does not affect histone hyperacetylation and DNA methylation levels at the ICE1 binding regions of the ZmDREB1 gene. Altogether, our findings indicate that HDACs positively regulate the expression of the cold-induced ZmDREB1 gene through histone modification and chromatin conformational changes and that this activation is both gene and site selective. PMID:21811564

A MYB/ZML Complex Regulates Wound-Induced Lignin Genes in Maize.

PubMed

Vélez-Bermúdez, Isabel-Cristina; Salazar-Henao, Jorge E; Fornalé, Silvia; López-Vidriero, Irene; Franco-Zorrilla, José-Manuel; Grotewold, Erich; Gray, John; Solano, Roberto; Schmidt, Wolfgang; Pagés, Montserrat; Riera, Marta; Caparros-Ruiz, David

2015-11-01

Lignin is an essential polymer in vascular plants that plays key structural roles in vessels and fibers. Lignification is induced by external inputs such as wounding, but the molecular mechanisms that link this stress to lignification remain largely unknown. In this work, we provide evidence that three maize (Zea mays) lignin repressors, MYB11, MYB31, and MYB42, participate in wound-induced lignification by interacting with ZML2, a protein belonging to the TIFY family. We determined that the three R2R3-MYB factors and ZML2 bind in vivo to AC-rich and GAT(A/C) cis-elements, respectively, present in a set of lignin genes. In particular, we show that MYB11 and ZML2 bind simultaneously to the AC-rich and GAT(A/C) cis-elements present in the promoter of the caffeic acid O-methyl transferase (comt) gene. We show that, like the R2R3-MYB factors, ZML2 also acts as a transcriptional repressor. We found that upon wounding and methyl jasmonate treatments, MYB11 and ZML2 proteins are degraded and comt transcription is induced. Based on these results, we propose a molecular regulatory mechanism involving a MYB/ZML complex in which wound-induced lignification can be achieved by the derepression of a set of lignin genes. © 2015 American Society of Plant Biologists. All rights reserved.

The Hippo effector Yorkie controls normal tissue growth by antagonizing scalloped-mediated default repression.

PubMed

Koontz, Laura M; Liu-Chittenden, Yi; Yin, Feng; Zheng, Yonggang; Yu, Jianzhong; Huang, Bo; Chen, Qian; Wu, Shian; Pan, Duojia

2013-05-28

The Hippo tumor suppressor pathway restricts tissue growth by inactivating the transcriptional coactivator Yki. Although Sd has been implicated as a DNA-binding transcription factor partner for Yki and can genetically account for gain-of-function Yki phenotypes, how Yki regulates normal tissue growth remains a long-standing puzzle because Sd, unlike Yki, is dispensable for normal growth in most Drosophila tissues. Here we show that the yki mutant phenotypes in multiple developmental contexts are rescued by inactivation of Sd, suggesting that Sd functions as a default repressor and that Yki promotes normal tissue growth by relieving Sd-mediated default repression. We further identify Tgi as a cofactor involved in Sd's default repressor function and demonstrate that the mammalian ortholog of Tgi potently suppresses the YAP oncoprotein in transgenic mice. These findings fill a major gap in Hippo-mediated transcriptional regulation and open up possibilities for modulating the YAP oncoprotein in cancer and regenerative medicine. Copyright © 2013 Elsevier Inc. All rights reserved.

Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

PubMed

Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

2015-04-28

A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

H-NS represses transcription of the flagellin gene lafA of lateral flagella in Vibrio parahaemolyticus.

PubMed

Wang, Yan; Zhang, Yiquan; Yin, Zhe; Wang, Jie; Zhu, Yongzhe; Peng, Haoran; Zhou, Dongsheng; Qi, Zhongtian; Yang, Wenhui

2018-01-01

Swarming motility is ultimately mediated by the proton-powered lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf genes is tightly regulated by a number of environmental conditions and regulatory factors. The nucleoid-associated DNA-binding protein H-NS is a small and abundant protein that is widely distributed in bacteria, and H-NS-like protein-dependent expression of laf genes has been identified in Vibrio cholerae and V. parahaemolyticus. The data presented here show that H-NS acts as a repressor of the swarming motility in V. parahaemolyticus. A single σ 28 -dependent promoter was detected for lafA encoding the flagellin of the lateral flagella, and its activity was directly repressed by H-NS. Thus, H-NS represses swarming motility by directly acting on lafA. Briefly, this work revealed a novel function for H-NS as a repressor of the expression of lafA and swarming motility in V. parahaemolyticus.

A novel E2 box-GATA element modulates Cdc6 transcription during human cells polyploidization

PubMed Central

Vilaboa, Nuria; Bermejo, Rodrigo; Martinez, Pilar; Bornstein, Rafael; Calés, Carmela

2004-01-01

Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements. PMID:15590906

A 44 bp intestine-specific hermaphrodite-specific enhancer from the C. elegans vit-2 vitellogenin gene is directly regulated by ELT-2, MAB-3, FKH-9 and DAF-16 and indirectly regulated by the germline, by daf-2/insulin signaling and by the TGF-β/Sma/Mab pathway.

PubMed

Goszczynski, Barbara; Captan, Vasile V; Danielson, Alicia M; Lancaster, Brett R; McGhee, James D

2016-05-01

The Caenorhabditis elegans vitellogenin genes are transcribed in the intestine of adult hermaphrodites but not of males. A 44-bp region from the vit-2 gene promoter is able largely to reconstitute this tissue-, stage- and sex-specific-expression. This "enhancer" contains a binding site for the DM-domain factor MAB-3, the male-specific repressor of vitellogenesis, as well as an activator site that we show is the direct target of the intestinal GATA factor ELT-2. We further show that the enhancer is directly activated by the winged-helix/forkhead-factor FKH-9, (whose gene has been shown by others to be a direct target of DAF-16), by an unknown activator binding to the MAB-3 site, and by the full C. elegans TGF-β/Sma/Mab pathway acting within the intestine. The vit-2 gene has been shown by others to be repressed by the daf-2/daf-16 insulin signaling pathway, which so strongly influences aging and longevity in C. elegans. We show that the activity of the 44 bp vit-2 enhancer is abolished by loss of daf-2 but is restored by simultaneous loss of daf-16. DAF-2 acts from outside of the intestine but DAF-16 acts both from outside of the intestine and from within the intestine where it binds directly to the same non-canonical target site that interacts with FKH-9. Activity of the 44 bp vit-2 enhancer is also inhibited by loss of the germline, in a manner that is only weakly influenced by DAF-16 but that is strongly influenced by KRI-1, a key downstream effector in the pathway by which germline loss increases C. elegans lifespan. The complex behavior of this enhancer presumably allows vitellogenin gene transcription to adjust to demands of body size, germline proliferation and nutritional state but we suggest that the apparent involvement of this enhancer in aging and longevity "pathways" could be incidental. Copyright © 2016 Elsevier Inc. All rights reserved.

The ELAV RNA-stability factor HuR binds the 5′-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation

PubMed Central

Meng, Zheng; King, Peter H.; Nabors, L. Burt; Jackson, Nateka L.; Chen, Ching-Yi; Emanuel, Peter D.; Blume, Scott W.

2005-01-01

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5′-untranslated region (5′-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5′-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3′-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5′-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5′-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5′-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5′-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5′-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis. PMID:15914670

Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning

PubMed Central

Sato'o, Yusuke; Hisatsune, Junzo; Nagasako, Yuria; Ono, Hisaya K.; Omoe, Katsuhiko

2015-01-01

We previously demonstrated the clonal complex 81 (CC81) subtype 1 lineage is the major staphylococcal food poisoning (SFP)-associated lineage in Japan (Y. Sato'o et al., J Clin Microbiol 52:2637–2640, 2014, http://dx.doi.org/10.1128/JCM.00661-14). Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP. PMID:26341202

Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning.

PubMed

Sato'o, Yusuke; Hisatsune, Junzo; Nagasako, Yuria; Ono, Hisaya K; Omoe, Katsuhiko; Sugai, Motoyuki

2015-11-01

We previously demonstrated the clonal complex 81 (CC81) subtype 1 lineage is the major staphylococcal food poisoning (SFP)-associated lineage in Japan (Y. Sato'o et al., J Clin Microbiol 52:2637-2640, 2014, http://dx.doi.org/10.1128/JCM.00661-14). Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Selection Shapes Transcriptional Logic and Regulatory Specialization in Genetic Networks.

PubMed

Fogelmark, Karl; Peterson, Carsten; Troein, Carl

2016-01-01

Living organisms need to regulate their gene expression in response to environmental signals and internal cues. This is a computational task where genes act as logic gates that connect to form transcriptional networks, which are shaped at all scales by evolution. Large-scale mutations such as gene duplications and deletions add and remove network components, whereas smaller mutations alter the connections between them. Selection determines what mutations are accepted, but its importance for shaping the resulting networks has been debated. To investigate the effects of selection in the shaping of transcriptional networks, we derive transcriptional logic from a combinatorially powerful yet tractable model of the binding between DNA and transcription factors. By evolving the resulting networks based on their ability to function as either a simple decision system or a circadian clock, we obtain information on the regulation and logic rules encoded in functional transcriptional networks. Comparisons are made between networks evolved for different functions, as well as with structurally equivalent but non-functional (neutrally evolved) networks, and predictions are validated against the transcriptional network of E. coli. We find that the logic rules governing gene expression depend on the function performed by the network. Unlike the decision systems, the circadian clocks show strong cooperative binding and negative regulation, which achieves tight temporal control of gene expression. Furthermore, we find that transcription factors act preferentially as either activators or repressors, both when binding multiple sites for a single target gene and globally in the transcriptional networks. This separation into positive and negative regulators requires gene duplications, which highlights the interplay between mutation and selection in shaping the transcriptional networks.

Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the mu opioid receptor (MOR) gene

PubMed Central

Kim, Chun Sung; Choi, Hack Sun; Hwang, Cheol Kyu; Song, Kyu Young; Lee, Byung-Kwon; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2006-01-01

Previously, we reported that the neuron-restrictive silencer element (NRSE) of mu opioid receptor (MOR) functions as a critical regulator to repress the MOR transcription in specific neuronal cells, depending on neuron-restriction silence factor (NRSF) expression levels [C.S.Kim, C.K.Hwang, H.S.Choi, K.Y.Song, P.Y.Law, L.N.Wei and H.H.Loh (2004) J. Biol. Chem., 279, 46464–46473]. Herein, we identify a conserved GC sequence next to NRSE region in the mouse MOR gene. The inhibition of Sp family factors binding to this GC box by mithramycin A led to a significant increase in the endogenous MOR transcription. In the co-immunoprecipitation experiment, NRSF interacted with the full-length Sp3 factor, but not with Sp1 or two short Sp3 isoforms. The sequence specific and functional binding by Sp3 at this GC box was confirmed by in vitro gel-shift assays using either in vitro translated proteins or nuclear extract, and by in vivo chromatin immunoprecipitation assays. Transient transfection assays showed that Sp3-binding site of the MOR gene is a functionally synergic repressor element with NRSE in NS20Y cells, but not in the NRSF negative PC12 cells. The results suggest that the synergic interaction between NRSF and Sp3 is required to negatively regulate MOR gene transcription and that transcription of MOR gene would be governed by the context of available transcription factors rather than by a master regulator. PMID:17130167

Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression

PubMed Central

Papadopoulos, Dimitrios K.; Reséndez-Pérez, Diana; Cárdenas-Chávez, Diana L.; Villanueva-Segura, Karina; Canales-del-Castillo, Ricardo; Felix, Daniel A.; Fünfschilling, Raphael; Gehring, Walter J.

2011-01-01

Segmental identity along the anteroposterior axis of bilateral animals is specified by Hox genes. These genes encode transcription factors, harboring the conserved homeodomain and, generally, a YPWM motif, which binds Hox cofactors and increases Hox transcriptional specificity in vivo. Here we derive synthetic Drosophila Antennapedia genes, consisting only of the YPWM motif and homeodomain, and investigate their functional role throughout development. Synthetic peptides and full-length Antennapedia proteins cause head-to-thorax transformations in the embryo, as well as antenna-to-tarsus and eye-to-wing transformations in the adult, thus converting the entire head to a mesothorax. This conversion is achieved by repression of genes required for head and antennal development and ectopic activation of genes promoting thoracic and tarsal fates, respectively. Synthetic Antennapedia peptides bind DNA specifically and interact with Extradenticle and Bric-à-brac interacting protein 2 cofactors in vitro and ex vivo. Substitution of the YPWM motif by alanines abolishes Antennapedia homeotic function, whereas substitution of YPWM by the WRPW repressor motif, which binds the transcriptional corepressor Groucho, allows all proteins to act as repressors only. Finally, naturally occurring variations in the size of the linker between the homeodomain and YPWM motif enhance Antennapedia repressive or activating efficiency, emphasizing the importance of linker size, rather than sequence, for specificity. Our results clearly show that synthetic Antennapedia genes are functional in vivo and therefore provide powerful tools for synthetic biology. Moreover, the YPWM motif is necessary—whereas the entire N terminus of the protein is dispensable—for Antennapedia homeotic function, indicating its dual role in transcriptional activation and repression by recruiting either coactivators or corepressors. PMID:21712439

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1).

PubMed

Fonseca, Bruno D; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M; Diao, Ilo T; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L; Hernández, Greco; Alain, Tommy; Damgaard, Christian K

2015-06-26

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1)*

PubMed Central

Fonseca, Bruno D.; Zakaria, Chadi; Jia, Jian-Jun; Graber, Tyson E.; Svitkin, Yuri; Tahmasebi, Soroush; Healy, Danielle; Hoang, Huy-Dung; Jensen, Jacob M.; Diao, Ilo T.; Lussier, Alexandre; Dajadian, Christopher; Padmanabhan, Niranjan; Wang, Walter; Matta-Camacho, Edna; Hearnden, Jaclyn; Smith, Ewan M.; Tsukumo, Yoshinori; Yanagiya, Akiko; Morita, Masahiro; Petroulakis, Emmanuel; González, Jose L.; Hernández, Greco; Alain, Tommy; Damgaard, Christian K.

2015-01-01

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5′TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1. PMID:25940091

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 developmentally arrested for several days before forming very small spore masses supported by a column of apparently undifferentiated cells. Thus, complete stalk cell differentiation appears to require at least two events: a commitment step, whereby the repression exerted by Dd-STATa is lifted, and a second step that is blocked in a Dd-STATa null organism. This latter step may involve extracellular cAMP, a known repressor of stalk cell differentiation, because Dd-STATa null cells are abnormally sensitive to the inhibitory effects of extracellular cyclic AMP.

The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during the first developmental cell lineage segregation.

PubMed

Huang, Daosheng; Guo, Guoji; Yuan, Ping; Ralston, Amy; Sun, Lingang; Huss, Mikael; Mistri, Tapan; Pinello, Luca; Ng, Huck Hui; Yuan, Guocheng; Ji, Junfeng; Rossant, Janet; Robson, Paul; Han, Xiaoping

2017-12-07

The first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and trophectoderm (TE). The transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4). However its downstream network in the developing embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify CDX2-regulated targets in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells. In addition, we examined the dynamics of gene expression changes using inducible CDX2 embryonic stem (ES) cells, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we identify putative novel regulatory elements that repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.

Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers

PubMed Central

Gao, Xuefei; Tsang, Jason C.H.; Gaba, Fortis; Wu, Donghai; Lu, Liming; Liu, Pentao

2014-01-01

The transcription activator–like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas9) utlilize distinct molecular mechanisms in targeting site recognition. The two proteins can be modified to carry additional functional domains to regulate expression of genomic loci in mammalian cells. In this study, we have compared the two systems in activation and suppression of the Oct4 and Nanog loci by targeting their enhancers. Although both are able to efficiently activate the luciferase reporters, the CRISPR/dCas9 system is much less potent in activating the endogenous loci and in the application of reprogramming somatic cells to iPS cells. Nevertheless, repression by CRISPR/dCas9 is comparable to or even better than TALE repressors. We demonstrated that dCas9 protein binding results in significant physical interference to binding of native transcription factors at enhancer, less efficient active histone markers induction or recruitment of activating complexes in gene activation. This study thus highlighted the merits and drawbacks of transcription regulation by each system. A combined approach of TALEs and CRISPR/dCas9 should provide an optimized solution to regulate genomic loci and to study genetic elements such as enhancers in biological processes including somatic cell reprogramming and guided differentiation. PMID:25223790

Post transcriptional control of the epigenetic stem cell regulator PLZF by sirtuin and HDAC deacetylases.

PubMed

McConnell, Melanie J; Durand, Laetitia; Langley, Emma; Coste-Sarguet, Lise; Zelent, Arthur; Chomienne, Christine; Kouzarides, Tony; Licht, Jonathan D; Guidez, Fabien

2015-01-01

The transcriptional repressor promyelocytic leukemia zinc finger protein (PLZF) is critical for the regulation of normal stem cells maintenance by establishing specific epigenetic landscape. We have previously shown that CBP/p300 acetyltransferase induces PLZF acetylation in order to increase its deoxynucleotidic acid (DNA) binding activity and to enhance its epigenetic function (repression of PLZF target genes). However, how PLZF is inactivated is not yet understood. In this study, we demonstrate that PLZF is deacetylated by both histone deacetylase 3 and the NAD+ dependent deacetylase silent mating type information regulation 2 homolog 1 (SIRT1). Unlike other PLZF-interacting deacetylases, these two proteins interact with the zinc finger domain of PLZF, where the activating CBP/p300 acetylation site was previously described, inducing deacetylation of lysines 647/650/653. Overexpression of histone deacetylase 3 (HDAC3) and SIRT1 is associated with loss of PLZF DNA binding activity and decreases PLZF transcriptional repression. As a result, the chromatin status of the promoters of PLZF target genes, involved in oncogenesis, shift from a heterochromatin to an open euchromatin environment leading to gene expression even in the presence of PLZF. Consequently, SIRT1 and HDAC3 mediated-PLZF deacetylation provides for rapid control and fine-tuning of PLZF activity through post-transcriptional modification to regulate gene expression and cellular homeostasis.

Structural basis of DNA target recognition by the B3 domain of Arabidopsis epigenome reader VAL1

PubMed Central

Sasnauskas, Giedrius; Kauneckaitė, Kotryna; Siksnys, Virginijus

2018-01-01

Abstract Arabidopsis thaliana requires a prolonged period of cold exposure during winter to initiate flowering in a process termed vernalization. Exposure to cold induces epigenetic silencing of the FLOWERING LOCUS C (FLC) gene by Polycomb group (PcG) proteins. A key role in this epigenetic switch is played by transcriptional repressors VAL1 and VAL2, which specifically recognize Sph/RY DNA sequences within FLC via B3 DNA binding domains, and mediate recruitment of PcG silencing machinery. To understand the structural mechanism of site-specific DNA recognition by VAL1, we have solved the crystal structure of VAL1 B3 domain (VAL1-B3) bound to a 12 bp oligoduplex containing the canonical Sph/RY DNA sequence 5′-CATGCA-3′/5′-TGCATG-3′. We find that VAL1-B3 makes H-bonds and van der Waals contacts to DNA bases of all six positions of the canonical Sph/RY element. In agreement with the structure, in vitro DNA binding studies show that VAL1-B3 does not tolerate substitutions at any position of the 5′-TGCATG-3′ sequence. The VAL1-B3–DNA structure presented here provides a structural model for understanding the specificity of plant B3 domains interacting with the Sph/RY and other DNA sequences. PMID:29660015

RBFOX2 protein domains and cellular activities.

PubMed

Arya, Anurada D; Wilson, David I; Baralle, Diana; Raponi, Michaela

2014-08-01

RBFOX2 (RNA-binding protein, Fox-1 homologue 2)/RBM9 (RNA-binding-motif protein 9)/RTA (repressor of tamoxifen action)/HNRBP2 (hexaribonucleotide-binding protein 2) encodes an RNA-binding protein involved in tissue specific alternative splicing regulation and steroid receptors transcriptional activity. Its ability to regulate specific splicing profiles depending on context has been related to different expression levels of the RBFOX2 protein itself and that of other splicing regulatory proteins involved in the shared modulation of specific genes splicing. However, this cannot be the sole explanation as to why RBFOX2 plays a widespread role in numerous cellular mechanisms from development to cell survival dependent on cell/tissue type. RBFOX2 isoforms with altered protein domains exist. In the present article, we describe the main RBFOX2 protein domains, their importance in the context of splicing and transcriptional regulation and we propose that RBFOX2 isoform distribution may play a fundamental role in RBFOX2-specific cellular effects.

Stimulatory and inhibitory mechanisms of slow muscle-specific myosin heavy chain gene expression in fish: Transient and transgenic analysis of torafugu MYH{sub M86-2} promoter in zebrafish embryos

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

Asaduzzaman, Md.; Kinoshita, Shigeharu, E-mail: akino@mail.ecc.u-tokyo.ac.jp; Bhuiyan, Sharmin Siddique

The myosin heavy chain gene, MYH{sub M86-2}, exhibited restricted expression in slow muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional mechanisms involved in its expression are still ambiguous. The present study is the first extensive analysis of slow muscle-specific MYH{sub M86-2} promoter in fish for identifying the cis-elements that are crucial for its expression. Combining both transient transfection and transgenic approaches, we demonstrated that the 2614 bp 5′-flanking sequences of MYH{sub M86-2} contain a sufficient promoter activity to drive gene expression specific to superficial slow muscle fibers. Bymore » cyclopamine treatment, we also demonstrated that the differentiation of such superficial slow muscle fibers depends on hedgehog signaling activity. The deletion analyses defined an upstream fragment necessary for repressing ectopic MYH{sub M86-2} expression in the fast muscle fibers. The transcriptional mechanism that prevents MYH{sub M86-2} expression in the fast muscle fibers is mediated through Sox6 binding elements. We also demonstrated that Sox6 may function as a transcriptional repressor of MYH{sub M86-2} expression. We further discovered that nuclear factor of activated T cells (NFAT) binding elements plays a key role and myocyte enhancer factor-2 (MEF2) binding elements participate in the transcriptional regulation of MYH{sub M86-2} expression. - Highlights: ► MYH{sub M86-2} is highly expressed in slow muscle fibers of torafugu embryos and larvae. ► MYH{sub M86-2} promoter activity depends on the hedgehog signaling. ► Sox6 binding elements inhibits MYH{sub M86-2} expression in fast muscle fibers. ► Sox6 elements function as transcriptional repressor of MYH{sub M86-2} promoter activity. ► NFAT and MEF2 binding elements play a key role for directing MYH{sub M86-2} expression.« less

Up-regulation of miR-95-3p in hepatocellular carcinoma promotes tumorigenesis by targeting p21 expression

PubMed Central

Ye, Jian; Yao, Yufeng; Song, Qixue; Li, Sisi; Hu, Zhenkun; Yu, Yubing; Hu, Changqing; Da, Xingwen; Li, Hui; Chen, Qiuyun; Wang, Qing K.

2016-01-01

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers. To elucidate new regulatory mechanisms for heptocarcinogenesis, we investigated the regulation of p21, a cyclin-dependent kinase (CDK) inhibitor encoded by CDKN1A, in HCC. The expression level of p21 is decreased with the progression of HCC. Luciferase assays with a luciferase-p21-3′ UTR reporter and its serial deletions identified a 15-bp repressor element at the 3′-UTR of CDKN1A, which contains a binding site for miR-95-3p. Mutation of the binding site eliminated the regulatory effect of miR-95-3p on p21 expression. Posttranscriptional regulation of p21 expression by miR-95-3p is mainly on the protein level (suppression of translation). Overexpression of miR-95-3p in two different HCC cell lines, HepG2 and SMMC7721, significantly promoted cell proliferation, cell cycle progression and cell migration, whereas a miR-95-3p specific inhibitor decreased cell proliferation, cell cycle progression and cell migration. The effects of miR-95-3p on cellular functions were rescued by overexpression of p21. Overexpression of miR-95-3p promoted cell proliferation and tumor growth in HCC xenograft mouse models. Expression of miR-95-3p was significantly higher in HCC samples than in adjacent non-cancerous samples. These results demonstrate that miR-95-3p is a potential new marker for HCC and regulates hepatocarcinogenesis by directly targeting CDKN1A/p21 expression. PMID:27698442

Human papillomavirus type 56 polymorphism in Canadian women with and without cervical lesions.

PubMed

Rodrigues-Coutlée, Catherine; Archambault, Jacques; Money, Deborah; Ramanakumar, Agnihotram V; Raboud, Janet; Hankins, Catherine; Koushik, Anita; Richardson, Harriet; Brassard, Paul; Franco, Eduardo L; Coutlée, Francois

2013-12-01

The genomic diversity of high-risk human papillomaviruses (HPV) has been associated with viral persistence and HPV-induced lesions. Studies on HPV56 persistence are still pending. To assess the association between HPV56 polymorphism and HPV56 persistence and presence of high-grade cervical intraepithelial neoplasia (CIN2,3) or cancer. HPV56-positive cervical specimens from 204 women selected from a total of 4669 participants recruited in 5 epidemiological studies (parent studies) were further analyzed by PCR-sequencing of the long control region (LCR). Of the 81 women followed prospectively in cohort studies who could be classified, 34 had persistent and 47 had transient HPV56 infections. Variant HPV56-LCR-MTL-21 was detected more frequently in persistent infections (52.9%, 95% CI: 36.7-68.6%) than in transient infections (25.5%, 95% CI: 15.1-39.4). Considering only women recruited in a cohort of women infected or at high risk for HIV infection, infection with variant HPV56-LCR-MTL-21 (OR=4.4, 95% CI: 1.3-14.5) was significantly associated with HPV56 persistence controlling in multivariate analysis for high risk HPV detection and HIV infection. A variation at nucleotide 7800 in HPV56-LCR-MTL-21 resulted in the loss of a binding site for Elf-1 embedded in one of the E2 binding sites, a potential activator or repressor of expression of the HPV genome. HPV56 polymorphism was not associated with CIN2,3 or cancer in women enrolled in cross-sectional and case-control studies. Polymorphism in HPV56 may influence the risk that infections with this type will persist. Copyright © 2013 Elsevier B.V. All rights reserved.

The transcription factor DREAM represses A20 and mediates inflammation

PubMed Central

Tiruppathi, Chinnaswamy; Soni, Dheeraj; Wang, Dong-Mei; Xue, Jiaping; Singh, Vandana; Thippegowda, Prabhakar B.; Cheppudira, Bopaiah P.; Mishra, Rakesh K.; DebRoy, Auditi; Qian, Zhijian; Bachmaier, Kurt; Zhao, Youyang; Christman, John W.; Vogel, Stephen M.; Ma, Averil; Malik, Asrar B.

2014-01-01

Here we show that the transcription-repressor DREAM binds to the A20 promoter to repress the expression of A20, the deubiquitinase suppressing inflammatory NF-κB signaling. DREAM-deficient (Dream−/−) mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, USF1 binding to the DRE-associated E-box domain activated A20 expression in response to inflammatory stimuli. These studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy in diseases such as acute lung injury associated with unconstrained NF-κB activity. PMID:24487321

Structure of the Polycomb Group protein PCGF1 (NSPC1) in complex with BCOR reveals basis for binding selectivity of PCGF homologs

PubMed Central

Junco, Sarah E.; Wang, Renjing; Gaipa, John C.; Taylor, Alexander B.; Schirf, Virgil; Gearhart, Micah D.; Bardwell, Vivian J.; Demeler, Borries; Hart, P. John; Kim, Chongwoo A.

2014-01-01

Summary Polycomb Group RING finger homologs (PCGF1, 2, 3, 4, 5 and 6) are critical components in the assembly of distinct Polycomb Repression Complex 1 (PRC1) related complexes. Here we identify a protein interaction domain in BCL6 co-repressor, BCOR, which binds the ubiquitin-like RAWUL domain of PCGF1 (NSPC1) and PCGF3 but not of PCGF2 (MEL18) or PCGF4 (BMI1). Because of the selective binding, we have named this domain PCGF Ub-like fold Discriminator (PUFD). The structure of BCOR PUFD bound to PCGF1 reveals 1. that PUFD binds to the same surfaces as observed for a different Polycomb Group RAWUL domain and 2. the ability of PUFD to discriminate among RAWULs stems from the identity of specific residues within these interaction surfaces. These data are the first to show the molecular basis for determining the binding preference for a PCGF homolog, which ultimately helps determine the identity of the larger PRC1-like assembly. PMID:23523425

Translation repression via modulation of the cytoplasmic poly(A)-binding protein in the inflammatory response

PubMed Central

Zhang, Xu; Chen, Xiaoli; Liu, Qiuying; Zhang, Shaojie; Hu, Wenqian

2017-01-01

Gene expression is precisely regulated during the inflammatory response to control infection and limit the detrimental effects of inflammation. Here, we profiled global mRNA translation dynamics in the mouse primary macrophage-mediated inflammatory response and identified hundreds of differentially translated mRNAs. These mRNAs’ 3’UTRs have enriched binding motifs for several RNA-binding proteins, which implies extensive translational regulatory networks. We characterized one such protein, Zfp36, as a translation repressor. Using primary macrophages from a Zfp36-V5 epitope tagged knock-in mouse generated by CRISPR/Cas9-mediated genome editing, we found that the endogenous Zfp36 directly interacts with the cytoplasmic poly(A)-binding protein. Importantly, this interaction is required for the translational repression of Zfp36’s target mRNAs in resolving inflammation. Altogether, these results uncovered critical roles of translational regulations in controlling appropriate gene expression during the inflammatory response and revealed a new biologically relevant molecular mechanism of translational repression via modulating the cytoplasmic poly(A)-binding protein. DOI: http://dx.doi.org/10.7554/eLife.27786.001 PMID:28635594

Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100

PubMed Central

Liu, Shouan; Kracher, Barbara; Ziegler, Jörg; Birkenbihl, Rainer P; Somssich, Imre E

2015-01-01

The Arabidopsis mutant wrky33 is highly susceptible to Botrytis cinerea. We identified >1680 Botrytis-induced WRKY33 binding sites associated with 1576 Arabidopsis genes. Transcriptional profiling defined 318 functional direct target genes at 14 hr post inoculation. Comparative analyses revealed that WRKY33 possesses dual functionality acting either as a repressor or as an activator in a promoter-context dependent manner. We confirmed known WRKY33 targets involved in hormone signaling and phytoalexin biosynthesis, but also uncovered a novel negative role of abscisic acid (ABA) in resistance towards B. cinerea 2100. The ABA biosynthesis genes NCED3 and NCED5 were identified as direct targets required for WRKY33-mediated resistance. Loss-of-WRKY33 function resulted in elevated ABA levels and genetic studies confirmed that WRKY33 acts upstream of NCED3/NCED5 to negatively regulate ABA biosynthesis. This study provides the first detailed view of the genome-wide contribution of a specific plant transcription factor in modulating the transcriptional network associated with plant immunity. DOI: http://dx.doi.org/10.7554/eLife.07295.001 PMID:26076231

The Elav-like protein HuR exerts translational control of viral internal ribosome entry sites

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

Rivas-Aravena, Andrea; Ramdohr, Pablo; Vallejos, Maricarmen

2009-09-30

The human embryonic-lethal abnormal vision (ELAV)-like protein, HuR, has been recently found to be involved in the regulation of protein synthesis. In this study we show that HuR participates in the translational control of the HIV-1 and HCV IRES elements. HuR functions as a repressor of HIV-1 IRES activity and acts as an activator of the HCV IRES. The effect of HuR was evaluated in three independent experimental systems, rabbit reticulocyte lysate, HeLa cells, and Xenopus laevis oocytes, using both overexpression and knockdown approaches. Furthermore, results suggest that HuR mediated regulation of HIV-1 and HCV IRESes does not require directmore » binding of the protein to the RNA nor does it need the nuclear translocation of the IRES-containing RNAs. Finally, we show that HuR has a negative impact on post-integration steps of the HIV-1 replication cycle. Thus, our observations yield novel insights into the role of HuR in the post-transcriptional regulation of HCV and HIV-1 gene expression.« less

SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

PubMed Central

Pagliarini, Vittoria; Pelosi, Laura; Bustamante, Maria Blaire; Nobili, Annalisa; Berardinelli, Maria Grazia; D’Amelio, Marcello; Musarò, Antonio

2015-01-01

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre–messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3′ splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model. PMID:26438828

Enhancer Activation by Pharmacologic Displacement of LSD1 from GFI1 Induces Differentiation in Acute Myeloid Leukemia.

PubMed

Maiques-Diaz, Alba; Spencer, Gary J; Lynch, James T; Ciceri, Filippo; Williams, Emma L; Amaral, Fabio M R; Wiseman, Daniel H; Harris, William J; Li, Yaoyong; Sahoo, Sudhakar; Hitchin, James R; Mould, Daniel P; Fairweather, Emma E; Waszkowycz, Bohdan; Jordan, Allan M; Smith, Duncan L; Somervaille, Tim C P

2018-03-27

Pharmacologic inhibition of LSD1 promotes blast cell differentiation in acute myeloid leukemia (AML) with MLL translocations. The assumption has been that differentiation is induced through blockade of LSD1's histone demethylase activity. However, we observed that rapid, extensive, drug-induced changes in transcription occurred without genome-wide accumulation of the histone modifications targeted for demethylation by LSD1 at sites of LSD1 binding and that a demethylase-defective mutant rescued LSD1 knockdown AML cells as efficiently as wild-type protein. Rather, LSD1 inhibitors disrupt the interaction of LSD1 and RCOR1 with the SNAG-domain transcription repressor GFI1, which is bound to a discrete set of enhancers located close to transcription factor genes that regulate myeloid differentiation. Physical separation of LSD1/RCOR1 from GFI1 is required for drug-induced differentiation. The consequent inactivation of GFI1 leads to increased enhancer histone acetylation within hours, which directly correlates with the upregulation of nearby subordinate genes. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells.

PubMed

Molnár, Viktor; Érsek, Barbara; Wiener, Zoltán; Tömböl, Zsófia; Szabó, Péter M; Igaz, Péter; Falus, András

2012-03-01

MicroRNAs provide an additional layer in the regulation of gene expression acting as repressors with several targets at the posttranscriptional level. This study describes microRNA expression patterns during differentiation and activation of mast cells. The expression levels of 567 different mouse miRNAs were compared by microarray between c-Kit+ committed progenitors, mucosal mast cells, resting and IgE-crosslinked BMMCs in vitro. The strongest upregulation of miR-132 upon IgE-mediated activation was validated in human cord blood-derived mast cells as well. HB-EGF growth factor also upregulated upon activation and was ranked high by more prediction algorithms. Co-transfection of miR-132 mimicking precursor and the 3'UTR of human Hbegf-containing luciferase vector proves that the predicted binding site is functional. In line with this, neutralization of miR-132 by anti-miR inhibitor leads to sustained production of HB-EGF protein in activated mast cells. Our data provide a novel example for negative regulation of a growth factor by an upregulated miRNA. © Springer Basel AG 2011

DAX1 suppresses FXR transactivity as a novel co-repressor

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

Li, Jin; Lu, Yan; Liu, Ruya

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 familymore » 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.« less

Transcriptional repressor foxl1 regulates central nervous system development by suppressing shh expression in zebra fish.

PubMed

Nakada, Chisako; Satoh, Shinya; Tabata, Yoko; Arai, Ken-ichi; Watanabe, Sumiko

2006-10-01

We identified zebra fish forkhead transcription factor l1 (zfoxl1) as a gene strongly expressed in neural tissues such as midbrain, hindbrain, and the otic vesicle at the early embryonic stage. Loss of the function of zfoxl1 effected by morpholino antisense oligonucleotide resulted in defects in midbrain and eye development, and in that of formation of the pectoral fins. Interestingly, ectopic expression of shh in the midbrain and elevated pax2a expression in the optic stalk were observed in foxl1 MO-injected embryos. In contrast, expression of pax6a, which is negatively regulated by shh, was suppressed in the thalamus and pretectum regions, supporting the idea of augmentation of the shh signaling pathway by suppression of foxl1. Expression of zfoxl1-EnR (repressing) rather than zfoxl1-VP16 (activating) resulted in a phenotype similar to that induced by foxl1-mRNA, suggesting that foxl1 may act as a transcriptional repressor of shh in zebra fish embryos. Supporting this notion, foxl1 suppressed isolated 2.7-kb shh promoter activity in PC12 cells, and the minimal region of foxl1 required for its transcriptional repressor activity showed strong homology with the groucho binding motif, which is found in genes encoding various homeodomain proteins. In view of all of our data taken together, we propose zfoxl1 to be a novel regulator of neural development that acts by suppressing shh expression.

Transcriptional Repressor foxl1 Regulates Central Nervous System Development by Suppressing shh Expression in Zebra Fish†

PubMed Central

Nakada, Chisako; Satoh, Shinya; Tabata, Yoko; Arai, Ken-ichi; Watanabe, Sumiko

2006-01-01

We identified zebra fish forkhead transcription factor l1 (zfoxl1) as a gene strongly expressed in neural tissues such as midbrain, hindbrain, and the otic vesicle at the early embryonic stage. Loss of the function of zfoxl1 effected by morpholino antisense oligonucleotide resulted in defects in midbrain and eye development, and in that of formation of the pectoral fins. Interestingly, ectopic expression of shh in the midbrain and elevated pax2a expression in the optic stalk were observed in foxl1 MO-injected embryos. In contrast, expression of pax6a, which is negatively regulated by shh, was suppressed in the thalamus and pretectum regions, supporting the idea of augmentation of the shh signaling pathway by suppression of foxl1. Expression of zfoxl1-EnR (repressing) rather than zfoxl1-VP16 (activating) resulted in a phenotype similar to that induced by foxl1-mRNA, suggesting that foxl1 may act as a transcriptional repressor of shh in zebra fish embryos. Supporting this notion, foxl1 suppressed isolated 2.7-kb shh promoter activity in PC12 cells, and the minimal region of foxl1 required for its transcriptional repressor activity showed strong homology with the groucho binding motif, which is found in genes encoding various homeodomain proteins. In view of all of our data taken together, we propose zfoxl1 to be a novel regulator of neural development that acts by suppressing shh expression. PMID:16980626

Structural dynamics of the lac repressor-DNA complex revealed by a multiscale simulation.

PubMed

Villa, Elizabeth; Balaeff, Alexander; Schulten, Klaus

2005-05-10

A multiscale simulation of a complex between the lac repressor protein (LacI) and a 107-bp-long DNA segment is reported. The complex between the repressor and two operator DNA segments is described by all-atom molecular dynamics; the size of the simulated system comprises either 226,000 or 314,000 atoms. The DNA loop connecting the operators is modeled as a continuous elastic ribbon, described mathematically by the nonlinear Kirchhoff differential equations with boundary conditions obtained from the coordinates of the terminal base pairs of each operator. The forces stemming from the looped DNA are included in the molecular dynamics simulations; the loop structure and the forces are continuously recomputed because the protein motions during the simulations shift the operators and the presumed termini of the loop. The simulations reveal the structural dynamics of the LacI-DNA complex in unprecedented detail. The multiple domains of LacI exhibit remarkable structural stability during the simulation, moving much like rigid bodies. LacI is shown to absorb the strain from the looped DNA mainly through its mobile DNA-binding head groups. Even with large fluctuating forces applied, the head groups tilt strongly and keep their grip on the operator DNA, while the remainder of the protein retains its V-shaped structure. A simulated opening of the cleft of LacI by 500-pN forces revealed the interactions responsible for locking LacI in the V-conformation.

Regulatory T cells facilitate the nuclear accumulation of inducible cAMP early repressor (ICER) and suppress nuclear factor of activated T cell c1 (NFATc1)

PubMed Central

Vaeth, Martin; Gogishvili, Tea; Bopp, Tobias; Klein, Matthias; Berberich-Siebelt, Friederike; Gattenloehner, Stefan; Avots, Andris; Sparwasser, Tim; Grebe, Nadine; Schmitt, Edgar; Hünig, Thomas; Serfling, Edgar; Bodor, Josef

2011-01-01

Inducible cAMP early repressor (ICER) is a transcriptional repressor, which, because of alternate promoter use, is generated from the 3′ region of the cAMP response modulator (Crem) gene. Its expression and nuclear occurrence are elevated by high cAMP levels in naturally occurring regulatory T cells (nTregs). Using two mouse models, we demonstrate that nTregs control the cellular localization of ICER/CREM, and thereby inhibit IL-2 synthesis in conventional CD4+ T cells. Ablation of nTregs in depletion of regulatory T-cell (DEREG) mice resulted in cytosolic localization of ICER/CREM and increased IL-2 synthesis upon stimulation. Direct contacts between nTregs and conventional CD4+ T cells led to nuclear accumulation of ICER/CREM and suppression of IL-2 synthesis on administration of CD28 superagonistic (CD28SA) Ab. In a similar way, nTregs communicated with B cells and induced the cAMP-driven nuclear localization of ICER/CREM. High levels of ICER suppressed the induction of nuclear factor of activated T cell c1 (Nfatc1) gene in T cells whose inducible Nfatc1 P1 promoter bears two highly conserved cAMP-responsive elements to which ICER/CREM can bind. These findings suggest that nTregs suppress T-cell responses by the cAMP-dependent nuclear accumulation of ICER/CREM and inhibition of NFATc1 and IL-2 induction. PMID:21262800

Characterization of cis-acting elements required for autorepression of the equine herpesvirus 1 IE gene

PubMed Central

Kim, Seongman; Dai, Gan; O’Callaghan, Dennis J.; Kim, Seong Kee

2012-01-01

The immediate-early protein (IEP), the major regulatory protein encoded by the IE gene of equine herpesvirus 1 (EHV-1), plays a crucial role as both transcription activator and repressor during a productive lytic infection. To investigate the mechanism by which the EHV-1 IEP inhibits its own promoter, IE promoter-luciferase reporter plasmids containing wild-type and mutant IEP-binding site (IEBS) were constructed and used for luciferase reporter assays. The IEP inhibited transcription from its own promoter in the presence of a consensus IEBS (5’-ATCGT-3’) located near the transcription initiation site but did not inhibit when the consensus sequence was deleted. To determine whether the distance between the TATA box and the IEBS affects transcriptional repression, the IEBS was displaced from the original site by the insertion of synthetic DNA sequences. Luciferase reporter assays revealed that the IEP is able to repress its own promoter when the IEBS is located within 26-bp from the TATA box. We also found that the proper orientation and position of the IEBS were required for the repression by the IEP. Interestingly, the level of repression was significantly reduced when a consensus TATA sequence was deleted from the promoter region, indicating that the IEP efficiently inhibits its own promoter in a TATA box-dependent manner. Taken together, these results suggest that the EHV-1 IEP delicately modulates autoregulation of its gene through the consensus IEBS that is near the transcription initiation site and the TATA box. PMID:22265772

Characterization of cis-acting elements required for autorepression of the equine herpesvirus 1 IE gene.

PubMed

Kim, Seongman; Dai, Gan; O'Callaghan, Dennis J; Kim, Seong Kee

2012-04-01

The immediate-early protein (IEP), the major regulatory protein encoded by the IE gene of equine herpesvirus 1 (EHV-1), plays a crucial role as both transcription activator and repressor during a productive lytic infection. To investigate the mechanism by which the EHV-1 IEP inhibits its own promoter, IE promoter-luciferase reporter plasmids containing wild-type and mutant IEP-binding site (IEBS) were constructed and used for luciferase reporter assays. The IEP inhibited transcription from its own promoter in the presence of a consensus IEBS (5'-ATCGT-3') located near the transcription initiation site but did not inhibit when the consensus sequence was deleted. To determine whether the distance between the TATA box and the IEBS affects transcriptional repression, the IEBS was displaced from the original site by the insertion of synthetic DNA sequences. Luciferase reporter assays revealed that the IEP is able to repress its own promoter when the IEBS is located within 26-bp from the TATA box. We also found that the proper orientation and position of the IEBS were required for the repression by the IEP. Interestingly, the level of repression was significantly reduced when a consensus TATA sequence was deleted from the promoter region, indicating that the IEP efficiently inhibits its own promoter in a TATA box-dependent manner. Taken together, these results suggest that the EHV-1 IEP delicately modulates autoregulation of its gene through the consensus IEBS that is near the transcription initiation site and the TATA box. Copyright © 2012. Published by Elsevier B.V.

A DNA-binding protein from Candida albicans that binds to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence of C. albicans.

PubMed

Ishii, N; Yamamoto, M; Lahm, H W; Iizumi, S; Yoshihara, F; Nakayama, H; Arisawa, M; Aoki, Y

1997-02-01

Electromobility shift assays with a DNA probe containing the Saccharomyces cerevisiae ENO1 RPG box identified a specific DNA-binding protein in total protein extracts of Candida albicans. The protein, named Rbf1p (RPG-box-binding protein 1), bound to other S. cerevisiae RPG boxes, although the nucleotide recognition profile was not completely the same as that of S. cerevisiae Rap 1p (repressor-activator protein 1), an RPG-box-binding protein. The repetitive sequence of the C. albicans chromosomal telomere also competed with RPG-box binding to Rbf1p. For further analysis, we purified Rbf1p 57,600-fold from C. albicans total protein extracts, raised mAbs against the purified protein and immunologically cloned the gene, whose ORF specified a protein of 527 aa. The bacterially expressed protein showed RPG-box-binding activity with the same profile as that of the purified one. The Rbf1p, containing two glutamine-rich regions that are found in many transcription factors, showed transcriptional activation capability in S. cerevisiae and was predominantly observed in nuclei. These results suggest that Rbf1p is a transcription factor with telomere-binding activity in C. albicans.

Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

PubMed Central

Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

2014-01-01

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

PubMed

Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

2014-02-01

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.

Drosophila C-terminal binding protein, dCtBP is required for sensory organ prepattern and sharpens proneural transcriptional activity of the GATA factor Pnr.

PubMed

Biryukova, Inna; Heitzler, Pascal

2008-11-01

The peripheral nervous system is required for animals to detect and to relay environmental stimuli to central nervous system for the information processing. In Drosophila, the precise spatial and temporal expression of two proneural genes achaete (ac) and scute (sc), is necessary for development of the sensory organs. Here we present an evidence that the transcription co-repressor, dCtBP acts as a negative regulator of sensory organ prepattern. Loss of dCtBP function mutant exhibits ectopic sensory organs, while overexpression of dCtBP results in a dramatic loss of sensory organs. These phenotypes are correlated with mis-emerging of sensory organ precursors and perturbated expression of proneural transcription activator Ac. Mammalian CtBP-1 was identified via interaction with the consensus motif PXDLSX(K/R) of adenovirus E1A oncoprotein. We demonstrated that dCtBP binds directly to PLDLS motif of Drosophila Friend of GATA-1 protein, U-shaped and sharpens the adult sensory organ development. Moreover, we found that dCtBP mediates multivalent interaction with the GATA transcriptional activator Pannier and acts as a direct co-repressor of the Pannier-mediated activation of proneural genes. We demonstrated that Pannier genetically interacts with dCtBP-interacting protein HDAC1, suggesting that the dCtBP-dependent regulation of Pannier activity could utilize a repressive mechanism involving alteration of local chromatine structure.

The Transcriptional Regulators NorG and MgrA Modulate Resistance to both Quinolones and β-Lactams in Staphylococcus aureus▿

PubMed Central

Truong-Bolduc, Que Chi; Hooper, David C.

2007-01-01

MgrA is a known regulator of the expression of several multidrug transporters in Staphylococcus aureus. We identified another regulator of multiple efflux pumps, NorG, by its ability, like that of MgrA, to bind specifically to the promoter of the gene encoding the NorA efflux pump. NorG is a member of the family of the GntR-like transcriptional regulators, and it binds specifically to the putative promoters of the genes encoding multidrug efflux pumps NorA, NorB, NorC, and AbcA. Overexpression of norG produces a threefold increase in norB transcripts associated with a fourfold increase in the level of resistance to quinolones. In contrast, disruption of norG produces no change in the level of transcripts of norA, norB, and norC but causes an increase of at least threefold in the transcript level of abcA, associated with a fourfold increase in resistance to methicillin, cefotaxime, penicillin G, and nafcillin. Overexpression of cloned abcA caused an 8- to 128-fold increase in the level of resistance to all four β-lactam antibiotics. Furthermore, MgrA and NorG have opposite effects on norB and abcA expression. MgrA acts as an indirect repressor for norB and a direct activator for abcA, whereas NorG acts as a direct activator for norB and a direct repressor for abcA. PMID:17277059

A Novel Pathway Links Oxidative Stress to Loss of Insulin Growth Factor-2 (IGF2) Imprinting through NF-κB Activation

PubMed Central

Yang, Bing; Wagner, Jennifer; Damaschke, Nathan; Yao, Tianyu; Wuerzberger-Davis, Shelly M.; Lee, Moon-Hee; Svaren, John; Miyamoto, Shigeki; Jarrard, David F.

2014-01-01

Genomic imprinting is the allele-specific expression of a gene based on parental origin. Loss of imprinting(LOI) of Insulin-like Growth Factor 2 (IGF2) during aging is important in tumorigenesis, yet the regulatory mechanisms driving this event are largely unknown. In this study oxidative stress, measured by increased NF-κB activity, induces LOI in both cancerous and noncancerous human prostate cells. Decreased expression of the enhancer-blocking element CCCTC-binding factor(CTCF) results in reduced binding of CTCF to the H19-ICR (imprint control region), a major factor in the allelic silencing of IGF2. This ICR then develops increased DNA methylation. Assays identify a recruitment of the canonical pathway proteins NF-κB p65 and p50 to the CTCF promoter associated with the co-repressor HDAC1 explaining gene repression. An IκBα super-repressor blocks oxidative stress-induced activation of NF-κB and IGF2 imprinting is maintained. In vivo experiments using IκBα mutant mice with continuous NF-κB activation demonstrate increased IGF2 LOI further confirming a central role for canonical NF-κB signaling. We conclude CTCF plays a central role in mediating the effects of NF-κB activation that result in altered imprinting both in vitro and in vivo. This novel finding connects inflammation found in aging prostate tissues with the altered epigenetic landscape. PMID:24558376

The CACCC-Binding Protein KLF3/BKLF Represses a Subset of KLF1/EKLF Target Genes and Is Required for Proper Erythroid Maturation In Vivo

PubMed Central

Funnell, Alister P. W.; Norton, Laura J.; Mak, Ka Sin; Burdach, Jon; Artuz, Crisbel M.; Twine, Natalie A.; Wilkins, Marc R.; Power, Carl A.; Hung, Tzong-Tyng; Perdomo, José; Koh, Philip; Bell-Anderson, Kim S.; Orkin, Stuart H.; Fraser, Stuart T.; Perkins, Andrew C.; Pearson, Richard C. M.

2012-01-01

The CACCC-box binding protein erythroid Krüppel-like factor (EKLF/KLF1) is a master regulator that directs the expression of many important erythroid genes. We have previously shown that EKLF drives transcription of the gene for a second KLF, basic Krüppel-like factor, or KLF3. We have now tested the in vivo role of KLF3 in erythroid cells by examining Klf3 knockout mice. KLF3-deficient adults exhibit a mild compensated anemia, including enlarged spleens, increased red pulp, and a higher percentage of erythroid progenitors, together with elevated reticulocytes and abnormal erythrocytes in the peripheral blood. Impaired erythroid maturation is also observed in the fetal liver. We have found that KLF3 levels rise as erythroid cells mature to become TER119+. Consistent with this, microarray analysis of both TER119− and TER119+ erythroid populations revealed that KLF3 is most critical at the later stages of erythroid maturation and is indeed primarily a transcriptional repressor. Notably, many of the genes repressed by KLF3 are also known to be activated by EKLF. However, the majority of these are not currently recognized as erythroid-cell-specific genes. These results reveal the molecular and physiological function of KLF3, defining it as a feedback repressor that counters the activity of EKLF at selected target genes to achieve normal erythropoiesis. PMID:22711990

The B-subdomain of the Xenopus laevis XFIN KRAB-AB domain is responsible for its weaker transcriptional repressor activity compared to human ZNF10/Kox1.

PubMed

Born, Nadine; Thiesen, Hans-Jürgen; Lorenz, Peter

2014-01-01

The Krüppel-associated box (KRAB) domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.

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

PubMed Central

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

2014-01-01

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

Parallel SCF adaptor capture proteomics reveals a role for SCFFBXL17 in NRF2 activation via BACH1 repressor turnover.

PubMed

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

2013-10-10

Modular cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of parallel adaptor capture (PAC) proteomics and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCF(FBXL17) in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

AES/GRG5: more than just a dominant-negative TLE/GRG family member.

PubMed

Beagle, Brandon; Johnson, Gail V W

2010-11-01

The human Transducin-like Enhancer of Split (TLE) and mouse homologue, Groucho gene-related protein (GRG), represent a family of conserved non-DNA binding transcriptional modulatory proteins divided into two subgroups based upon size. The long TLE/GRGs consist of four pentadomain proteins that are dedicated co-repressors for multiple transcription factors (TF). The second TLE/GRG subgroup is composed of the Amino-terminal Enhancer of Split (AES) in humans and its mouse homolog GRG5 (AES/GRG5). In contrast to the dedicated co-repressor function of long TLE/GRGs, AES/GRG5 can both positively or negatively modulate various TF as well as non-TF proteins in a long TLE/GRG-dependent or -independent manner. Therefore, AES/GRG5 is a functionally dynamic protein that is not exclusively defined by its role as a long TLE/GRG antagonist. AES/GRG5 may function in various developmental and pathological processes but the functional characteristics of endogenous AES/GRG5 in a physiologically relevant context remains to be determined. © 2010 Wiley-Liss, Inc.

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR.

PubMed

Resch, Marcus; Striegl, Harald; Henssler, Eva Maria; Sevvana, Madhumati; Egerer-Sieber, Claudia; Schiltz, Emile; Hillen, Wolfgang; Muller, Yves A

2008-08-01

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder-order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates.

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. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The molecular mechanism of SPOROCYTELESS/NOZZLE in controlling Arabidopsis ovule development

PubMed Central

Wei, Baoye; Zhang, Jinzhe; Pang, Changxu; Yu, Hao; Guo, Dongshu; Jiang, Hao; Ding, Mingxin; Chen, Zhuoyao; Tao, Qing; Gu, Hongya; Qu, Li-Jia; Qin, Genji

2015-01-01

Ovules are essential for plant reproduction and develop into seeds after fertilization. SPOROCYTELESS/NOZZLE (SPL/NZZ) has been known for more than 15 years as an essential factor for ovule development in Arabidopsis, but the biochemical nature of SPL function has remained unsolved. Here, we demonstrate that SPL functions as an adaptor-like transcriptional repressor. We show that SPL recruits TOPLESS/TOPLESS-RELATED (TPL/TPR) co-repressors to inhibit the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors. We reveal that SPL uses its EAR motif at the C-terminal end to recruit TPL/TPRs and its N-terminal part to bind and inhibit the TCPs. We demonstrate that either disruption of TPL/TPRs or overexpression of TCPs partially phenocopies the defects of megasporogenesis in spl. Moreover, disruption of TCPs causes phenotypes that resemble spl-D gain-of-function mutants. These results define the action mechanism for SPL, which along with TPL/TPRs controls ovule development by repressing the activities of key transcription factors. Our findings suggest that a similar gene repression strategy is employed by both plants and fungi to control sporogenesis. PMID:25378179

Yin Yang 1 Is a Critical Repressor of Matrix Metalloproteinase-9 Expression in Brain Neurons*

PubMed Central

Rylski, Marcin; Amborska, Renata; Zybura, Katarzyna; Mioduszewska, Barbara; Michaluk, Piotr; Jaworski, Jacek; Kaczmarek, Leszek

2008-01-01

Membrane depolarization controls long lasting adaptive neuronal changes in brain physiology and pathology. Such responses are believed to be gene expression-dependent. Notably, however, only a couple of gene repressors active in nondepolarized neurons have been described. In this study, we show that in the unstimulated rat hippocampus in vivo, as well as in the nondepolarized brain neurons in primary culture, the transcriptional regulator Yin Yang 1 (YY1) is bound to the proximal Mmp-9 promoter and strongly represses Mmp-9 transcription. Furthermore, we demonstrate that monoubiquitinated and CtBP1 (C-terminal binding protein 1)-bound YY1 regulates Mmp-9 mRNA synthesis in rat brain neurons controlling its transcription apparently via HDAC3-dependent histone deacetylation. In conclusion, our data suggest that YY1 exerts, via epigenetic mechanisms, a control over neuronal expression of MMP-9. Because MMP-9 has recently been shown to play a pivotal role in physiological and pathological neuronal plasticity, YY1 may be implicated in these phenomena as well. PMID:18940814

The organization of RNA contacts by PTB for regulation of FAS splicing

PubMed Central

Mickleburgh, Ian; Kafasla, Panagiota; Cherny, Dmitry; Llorian, Miriam; Curry, Stephen; Jackson, Richard J.; Smith, Christopher W.J.

2014-01-01

Post-transcriptional steps of gene expression are regulated by RNA binding proteins. Major progress has been made in characterizing RNA-protein interactions, from high resolution structures to transcriptome-wide profiling. Due to the inherent technical challenges, less attention has been paid to the way in which proteins with multiple RNA binding domains engage with target RNAs. We have investigated how the four RNA recognition motif (RRM) domains of Polypyrimidine tract binding (PTB) protein, a major splicing regulator, interact with FAS pre-mRNA under conditions in which PTB represses FAS exon 6 splicing. A combination of tethered hydroxyl radical probing, targeted inactivation of individual RRMs and single molecule analyses revealed an unequal division of labour between the four RRMs of PTB. RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA. The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker. Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained. PMID:24957602

Selection Shapes Transcriptional Logic and Regulatory Specialization in Genetic Networks

PubMed Central

Fogelmark, Karl; Peterson, Carsten; Troein, Carl

2016-01-01

Background Living organisms need to regulate their gene expression in response to environmental signals and internal cues. This is a computational task where genes act as logic gates that connect to form transcriptional networks, which are shaped at all scales by evolution. Large-scale mutations such as gene duplications and deletions add and remove network components, whereas smaller mutations alter the connections between them. Selection determines what mutations are accepted, but its importance for shaping the resulting networks has been debated. Methodology To investigate the effects of selection in the shaping of transcriptional networks, we derive transcriptional logic from a combinatorially powerful yet tractable model of the binding between DNA and transcription factors. By evolving the resulting networks based on their ability to function as either a simple decision system or a circadian clock, we obtain information on the regulation and logic rules encoded in functional transcriptional networks. Comparisons are made between networks evolved for different functions, as well as with structurally equivalent but non-functional (neutrally evolved) networks, and predictions are validated against the transcriptional network of E. coli. Principal Findings We find that the logic rules governing gene expression depend on the function performed by the network. Unlike the decision systems, the circadian clocks show strong cooperative binding and negative regulation, which achieves tight temporal control of gene expression. Furthermore, we find that transcription factors act preferentially as either activators or repressors, both when binding multiple sites for a single target gene and globally in the transcriptional networks. This separation into positive and negative regulators requires gene duplications, which highlights the interplay between mutation and selection in shaping the transcriptional networks. PMID:26927540

Dynamic pre-BCR homodimers fine-tune autonomous survival signals in B cell precursor acute lymphoblastic leukemia

PubMed Central

Erasmus, M. Frank; Matlawska-Wasowska, Ksenia; Kinjyo, Ichiko; Mahajan, Avanika; Winter, Stuart S.; Xu, Li; Horowitz, Michael; Lidke, Diane S.; Wilson, Bridget S.

2017-01-01

The pre-B cell receptor (pre-BCR) is an immature form of the BCR critical for early B lymphocyte development. It is composed of the membrane-bound immunoglobulin (Ig) heavy chain, surrogate light chain components, and the signaling subunits Igα and Igβ. We developed monovalent Quantum Dot (QD)-labeled probes specific for Igβ to study the behavior of pre-BCRs engaged in autonomous, ligand-independent signaling in live B cells. Single-particle tracking revealed that QD-labeled pre-BCRs engaged in transient, but frequent, homotypic interactions. Receptor motion was correlated at short separation distances, consistent with the formation of dimers and higher-order oligomers. Repeated encounters between diffusing pre-BCRs appeared to reflect transient co-confinement in plasma membrane domains. In human B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, we showed that frequent, short-lived, homotypic pre-BCR interactions stimulated survival signals, including expression of BCL6, which encodes a transcriptional repressor. These survival signals were blocked by inhibitory monovalent antigen-binding antibody fragments (Fabs) specific for the surrogate light chain components of the pre-BCR or by inhibitors of the tyrosine kinases Lyn and Syk. For comparison, we evaluated pre-BCR aggregation mediated by dimeric galectin-1, which has binding sites for carbohydrate and for the λ5 component of the surrogate light chain. Galectin-1 binding resulted in the formation of large, highly immobile pre-BCR aggregates, which was partially relieved by the addition of lactose to prevent the crosslinking of galectin-BCR complexes to other glycosylated membrane components. Analysis of the pre-BCR and its signaling partners suggested that they could be potential targets for combination therapy in BCP-ALL. PMID:27899526

Folding pathway of the pyridoxal 5′-phosphate C-S lyase MalY from Escherichia coli

PubMed Central

2005-01-01

MalY from Escherichia coli is a bifunctional dimeric PLP (pyridoxal 5′-phosphate) enzyme acting as a β-cystathionase and as a repressor of the maltose system. The spectroscopic and molecular properties of the holoenzyme, in the untreated and NaBH4-treated forms, and of the apoenzyme have been elucidated. A systematic study of the urea-induced unfolding of MalY has been monitored by gel filtration, cross-linking, ANS (8-anilino-1-naphthalenesulphonic acid) binding and by visible, near- and far-UV CD, fluorescence and NMR spectroscopies under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M urea, gives rise to a partially active dimeric species that binds PLP. The second equilibrium transition involving dimer dissociation, release of PLP and loss of lyase activity leads to the formation of a monomeric equilibrium intermediate. It is a partially unfolded molecule that retains most of the native-state secondary structure, binds significant amounts of ANS (a probe for exposed hydrophobic surfaces) and tends to self-associate. The self-associated aggregates predominate at urea concentrations of 2–4 M for holoMalY. The third step represents the complete unfolding of the enzyme. These results when compared with the urea-induced unfolding profiles of apoMalY and NaBH4-reduced holoenzyme suggest that the coenzyme group attached to the active-site lysine residue increases the stability of the dimeric enzyme. Both holo- and apo-MalY could be successfully refolded into the active enzyme with an 85% yield. Further refolding studies suggest that large misfolded soluble aggregates that cannot be refolded could be responsible for the incomplete re-activation. PMID:15823094

ArgR of Streptomyces coelicolor Is a Pleiotropic Transcriptional Regulator: Effect on the Transcriptome, Antibiotic Production, and Differentiation in Liquid Cultures

PubMed Central

Botas, Alma; Pérez-Redondo, Rosario; Rodríguez-García, Antonio; Álvarez-Álvarez, Rubén; Yagüe, Paula; Manteca, Angel; Liras, Paloma

2018-01-01

ArgR is a well-characterized transcriptional repressor controlling the expression of arginine and pyrimidine biosynthetic genes in bacteria. In this work, the biological role of Streptomyces coelicolor ArgR was analyzed by comparing the transcriptomes of S. coelicolor ΔargR and its parental strain, S. coelicolor M145, at five different times over a 66-h period. The effect of S. coelicolor ArgR was more widespread than that of the orthologous protein of Escherichia coli, affecting the expression of 1544 genes along the microarray time series. This S. coelicolor regulator repressed the expression of arginine and pyrimidine biosynthetic genes, but it also modulated the expression of genes not previously described to be regulated by ArgR: genes involved in nitrogen metabolism and nitrate utilization; the act, red, and cpk genes for antibiotic production; genes for the synthesis of the osmotic stress protector ectoine; genes related to hydrophobic cover formation and sporulation (chaplins, rodlins, ramR, and whi genes); all the cwg genes encoding proteins for glycan cell wall biosynthesis; and genes involved in gas vesicle formation. Many of these genes contain ARG boxes for ArgR binding. ArgR binding to seven new ARG boxes, located upstream or near the ectA-ectB, afsS, afsR, glnR, and redH genes, was tested by DNA band-shift assays. These data and those of previously assayed fragments permitted the construction of an improved model of the ArgR binding site. Interestingly, the overexpression of sporulation genes observed in the ΔargR mutant in our culture conditions correlated with a sporulation-like process, an uncommon phenotype. PMID:29545785

NF-κB Regulation of YY1 Inhibits Skeletal Myogenesis through Transcriptional Silencing of Myofibrillar Genes▿ †

PubMed Central

Wang, Huating; Hertlein, Erin; Bakkar, Nadine; Sun, Hao; Acharyya, Swarnali; Wang, Jingxin; Carathers, Micheal; Davuluri, Ramana; Guttridge, Denis C.

2007-01-01

NF-κB signaling is implicated as an important regulator of skeletal muscle homeostasis, but the mechanisms by which this transcription factor contributes to muscle maturation and turnover remain unclear. To gain insight into these mechanisms, gene expression profiling was examined in C2C12 myoblasts devoid of NF-κB activity. Interestingly, even in proliferating myoblasts, the absence of NF-κB caused the pronounced induction of several myofibrillar genes, suggesting that NF-κB functions as a negative regulator of late-stage muscle differentiation. Although several myofibrillar promoters contain predicted NF-κB binding sites, functional analysis using the troponin-I2 gene as a model revealed that NF-κB-mediated repression does not occur through direct DNA binding. In the search for an indirect mediator, the transcriptional repressor YinYang1 (YY1) was identified. While inducers of NF-κB stimulated YY1 expression in multiple cell types, genetic ablation of the RelA/p65 subunit of NF-κB in both cultured cells and adult skeletal muscle correlated with reduced YY1 transcripts and protein. NF-κB regulation of YY1 occurred at the transcriptional level, mediated by direct binding of the p50/p65 heterodimer complex to the YY1 promoter. Furthermore, YY1 was found associated with multiple myofibrillar promoters in C2C12 myoblasts containing NF-κB activity. Based on these results, we propose that NF-κB regulation of YY1 and transcriptional silencing of myofibrillar genes represent a new mechanism by which NF-κB functions in myoblasts to modulate skeletal muscle differentiation. PMID:17438126

The eIF4E-binding proteins are modifiers of cytoplasmic eIF4E relocalization during the heat shock response.

PubMed

Sukarieh, R; Sonenberg, N; Pelletier, J

2009-05-01

Stress granules (SGs) arise as a consequence of cellular stress, contain stalled translation preinitiation complexes, and are associated with cell survival during environmental insults. SGs are dynamic entities with proteins relocating into and out of them during stress. Among the repertoire of proteins present in SGs is eukaryotic initiation factor 4E (eIF4E), a translation factor required for cap-dependent translation and that regulates a rate-limiting step for protein synthesis. Herein, we demonstrate that localization of eIF4E to SGs is dependent on the presence of a family of repressor proteins, eIF4E-binding proteins (4E-BPs). Our results demonstrate that 4E-BPs regulate the SG localization of eIF4E.

Evidence for the negative regulation of phytase gene expression in Streptomyces lividans and Streptomyces coelicolor.

PubMed

Boukhris, Ines; Dulermo, Thierry; Chouayekh, Hichem; Virolle, Marie-Joëlle

2016-01-01

Sco7697, a gene encoding a phytase, enzyme able to degrade phytate (myo-inositol 1,2,3,4,5,6-hexakis phosphate), the most abundant phosphorus storing compound in plants is present in the genome of S. coelicolor, a soil born bacteria with a saprophytic lifestyle. The expression of this gene was previously shown to be induced in conditions of Pi limitation by the response regulator PhoP binding to an operator sequence, the PHO box, located upstream of the -35 promoter sequence. A close examination of the promoter region of sco7697 revealed the presence of another putative operator site, a Direct Repeat (DR), located downstream of the -10 promoter sequence. In order to determine whether this DR played a role in regulation of sco7697 expression, different variants of the phytase gene promoter region were transcriptionally fused to the ß-glucuronidase reporter gene (GUS). As expected, deletion of the PHO box led to abolition of sco7697 induction in conditions of Pi limitation. Interestingly, alteration of the DR correlated with a dramatic increase of GUS expression but only when PhoP was present. These results demonstrated that this DR is the site of strong negative regulation by an unknown repressor. The latter would impede the necessary activation of phytase expression by PhoP. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Key Evolutionary Mutation Enhances DNA Binding of the FOXP2 Forkhead Domain.

PubMed

Morris, Gavin; Fanucchi, Sylvia

2016-04-05

Forkhead box (FOX) transcription factors share a conserved forkhead DNA binding domain (FHD) and are key role players in the development of many eukaryotic species. Their involvement in various congenital disorders and cancers makes them clinically relevant targets for novel therapeutic strategies. Among them, the FOXP subfamily of multidomain transcriptional repressors is unique in its ability to form DNA binding homo and heterodimers. The truncated FOXP2 FHD, in the absence of the leucine zipper, exists in equilibrium between monomeric and domain-swapped dimeric states in vitro. As a consequence, determining the DNA binding properties of the FOXP2 FHD becomes inherently difficult. In this work, two FOXP2 FHD hinge loop mutants have been generated to successfully prevent both the formation (A539P) and the dissociation (F541C) of the homodimers. This allows for the separation of the two species for downstream DNA binding studies. Comparison of DNA binding of the different species using electrophoretic mobility shift assay, fluorescence anisotropy and isothermal titration calorimetry indicates that the wild-type FOXP2 FHD binds DNA as a monomer. However, comparison of the DNA-binding energetics of the monomer and wild-type FHD, reveals that there is a difference in the mechanism of binding between the two species. We conclude that the naturally occurring reverse mutation (P539A) seen in the FOXP subfamily increases DNA binding affinity and may increase the potential for nonspecific binding compared to other FOX family members.

Promoter/repressor system of Lactobacillus plantarum phage og1e: characterization of the promoters pR49-pR-pL and overproduction of the cro-like protein cng in Escherichia coli.

PubMed

Kakikawa, M; Watanabe, N; Funawatashi, T; Oki, M; Yasukawa, H; Taketo, A; Kodaira, K I

1998-07-30

The Lactobacillus plantarum phage og1e (42259bp) has two repressor-like genes cng and cpg oriented oppositely, accompanied by three potential promoters pR, pL and pR49, and seven operator-like sequences (GATAC-boxes) (Kodaira et al., 1997). In this study, the og1e putative promoters were introduced into the Escherichia coli promoter-detecting plasmid pKK232-8. In E. coli CK111, pR (pKPR1), pL (pKPL1) and pR49 (pKPR49) exhibited distinct CAT activities. When pKPR1 or pKPL1 was coexistent with a compatible plasmid pACYC184 carrying pR-cng (pA4PRCN1), the CAT activity was decreased significantly. On the other hand, cng directed a protein (Cng) of 10.1 kDa in E. coli under the control of T7 promoter. Gel mobility-shift assays demonstrated that Cng binds specifically to a DNA region containing the GATAC-boxes. In addition, primer extension analyses demonstrated that the two sequences pR and pL act as a promoter in L. plantarum as well as in E. coli. These results suggested that the potential promoters pR and pL probably function for the lytic and lysogenic pathways, respectively, and Cng may act as a repressor presumably through the GATAC-boxes as operators.

Left-right axis asymmetry determining human Cryptic gene is transcriptionally repressed by Snail.

PubMed

Gupta, Kartik; Pilli, Vijaya Satish Sekhar; Aradhyam, Gopala Krishna

2016-10-28

Establishment of the left-right axis is important for positioning organs asymmetrically in the developing vertebrate-embryo. A number of factors like maternally deposited molecules have emerged essential in initiating the specification of the axis; the downstream events, however, are regulated by signal-transduction and gene-expression changes identifying which remains a crucial challenge. The EGF-CFC family member Cryptic, that functions as a co-receptor for some TGF-beta ligands, is developmentally expressed in higher mammals and mutations in the gene cause loss or change in left-right axis asymmetry. Despite the strong phenotype, no transcriptional-regulator of this gene is known till date. Using promoter-analyses tools, we found strong evidence that the developmentally essential transcription factor Snail binds to the human Cryptic-promoter. We cloned the promoter-region of human Cryptic in a reporter gene and observed decreased Cryptic-promoter activation upon increasing Snail expression. Further, the expression of Cryptic is down-regulated upon exogenous Snail expression, validating the reporter assays and the previously identified role of Snail as a transcriptional repressor. Finally, we demonstrate using gel-shift assay that Snail in nuclear extract of PANC1 cells interacts with the promoter-construct bearing putative Snail binding sites and confirm this finding using chromatin immunoprecipitation assay. Snail represses the expression of human Cryptic and therefore, might affect the signaling via Nodal that has previously been demonstrated to specify the left-right axis using the EGF-CFC co-receptors.

Transcriptional regulation of NAD metabolism in bacteria: genomic reconstruction of NiaR (YrxA) regulon

PubMed Central

Rodionov, Dmitry A.; Li, Xiaoqing; Rodionova, Irina A.; Yang, Chen; Sorci, Leonardo; Dervyn, Etienne; Martynowski, Dariusz; Zhang, Hong; Gelfand, Mikhail S.; Osterman, Andrei L.

2008-01-01

A comparative genomic approach was used to reconstruct transcriptional regulation of NAD biosynthesis in bacteria containing orthologs of Bacillus subtilis gene yrxA, a previously identified niacin-responsive repressor of NAD de novo synthesis. Members of YrxA family (re-named here NiaR) are broadly conserved in the Bacillus/Clostridium group and in the deeply branching Fusobacteria and Thermotogales lineages. We analyzed upstream regions of genes associated with NAD biosynthesis to identify candidate NiaR-binding DNA motifs and assess the NiaR regulon content in these species. Representatives of the two distinct types of candidate NiaR-binding sites, characteristic of the Firmicutes and Thermotogales, were verified by an electrophoretic mobility shift assay. In addition to transcriptional control of the nadABC genes, the NiaR regulon in some species extends to niacin salvage (the pncAB genes) and includes uncharacterized membrane proteins possibly involved in niacin transport. The involvement in niacin uptake proposed for one of these proteins (re-named NiaP), encoded by the B. subtilis gene yceI, was experimentally verified. In addition to bacteria, members of the NiaP family are conserved in multicellular eukaryotes, including human, pointing to possible NaiP involvement in niacin utilization in these organisms. Overall, the analysis of the NiaR and NrtR regulons (described in the accompanying paper) revealed mechanisms of transcriptional regulation of NAD metabolism in nearly a hundred diverse bacteria. PMID:18276644

An InDel in the Promoter of Al-ACTIVATED MALATE TRANSPORTER9 Selected during Tomato Domestication Determines Fruit Malate Contents and Aluminum Tolerance[OPEN

PubMed Central

Wang, Xin; Hu, Tixu; Zhang, Fengxia; Wang, Bing; Li, Changxin; Yang, Tianxia; Li, Hanxia; Lu, Yongen; Ye, Zhibiao

2017-01-01

Deciphering the mechanism of malate accumulation in plants would contribute to a greater understanding of plant chemistry, which has implications for improving flavor quality in crop species and enhancing human health benefits. However, the regulation of malate metabolism is poorly understood in crops such as tomato (Solanum lycopersicum). Here, we integrated a metabolite-based genome-wide association study with linkage mapping and gene functional studies to characterize the genetics of malate accumulation in a global collection of tomato accessions with broad genetic diversity. We report that TFM6 (tomato fruit malate 6), which corresponds to Al-ACTIVATED MALATE TRANSPORTER9 (Sl-ALMT9 in tomato), is the major quantitative trait locus responsible for variation in fruit malate accumulation among tomato genotypes. A 3-bp indel in the promoter region of Sl-ALMT9 was linked to high fruit malate content. Further analysis indicated that this indel disrupts a W-box binding site in the Sl-ALMT9 promoter, which prevents binding of the WRKY transcription repressor Sl-WRKY42, thereby alleviating the repression of Sl-ALMT9 expression and promoting high fruit malate accumulation. Evolutionary analysis revealed that this highly expressed Sl-ALMT9 allele was selected for during tomato domestication. Furthermore, vacuole membrane-localized Sl-ALMT9 increases in abundance following Al treatment, thereby elevating malate transport and enhancing Al resistance. PMID:28814642

Key role of an ADP - ribose - dependent transcriptional regulator of NAD metabolism for fitness and virulence of Pseudomonas aeruginosa.

PubMed

Okon, Elza; Dethlefsen, Sarah; Pelnikevich, Anna; Barneveld, Andrea van; Munder, Antje; Tümmler, Burkhard

2017-01-01

NAD is an essential co-factor of redox reactions and metabolic conversions of NAD-dependent enzymes. NAD biosynthesis in the opportunistic pathogen Pseudomonas aeruginosa has yet not been experimentally explored. The in silico search for orthologs in the P. aeruginosa PAO1 genome identified the operon pncA - pncB1-nadE (PA4918-PA4920) to encode the nicotinamidase, nicotinate phosporibosyltransferase and Nad synthase of salvage pathway I. The functional role of the preceding genes PA4917 and PA4916 was resolved by the characterization of recombinant protein. PA4917 turned out to encode the nicotinate mononucleotide adenylyltransferase NadD2 and PA4916 was determined to encode the transcriptional repressor NrtR that binds to an intergenic sequence between nadD2 and pncA. Complex formation between the catalytically inactive Nudix protein NrtR and its DNA binding site was suppressed by the antirepressor ADP-ribose. NrtR plasposon mutagenesis abrogated virulence of P. aeruginosa TBCF10839 in a murine acute airway infection model and constrained its metabolite profile. When grown together with other isogenic plasposon mutants, the nrtR knock-out was most compromised in competitive fitness to persist in nutrient-rich medium in vitro or murine airways in vivo. This example demonstrates how tightly metabolism and virulence can be intertwined by key elements of metabolic control. Copyright © 2016 Elsevier GmbH. All rights reserved.

Effect of Rap1 binding on DNA distortion and potassium permanganate hypersensitivity.

PubMed

Le Bihan, Yann-Vaï; Matot, Béatrice; Pietrement, Olivier; Giraud-Panis, Marie-Josèphe; Gasparini, Sylvaine; Le Cam, Eric; Gilson, Eric; Sclavi, Bianca; Miron, Simona; Le Du, Marie-Hélène

2013-03-01

Repressor activator protein 1 (Rap1) is an essential factor involved in transcription and telomere stability in the budding yeast Saccharomyces cerevisiae. Its interaction with DNA causes hypersensitivity to potassium permanganate, suggesting local DNA melting and/or distortion. In this study, various Rap1-DNA crystal forms were obtained using specifically designed crystal screens. Analysis of the DNA conformation showed that its distortion was not sufficient to explain the permanganate reactivity. However, anomalous data collected at the Mn edge using a Rap1-DNA crystal soaked in potassium permanganate solution indicated that the DNA conformation in the crystal was compatible with interaction with permanganate ions. Sequence-conservation analysis revealed that double-Myb-containing Rap1 proteins all carry a fully conserved Arg580 at a position that may favour interaction with permanganate ions, although it is not involved in the hypersensitive cytosine distortion. Permanganate reactivity assays with wild-type Rap1 and the Rap1[R580A] mutant demonstrated that Arg580 is essential for hypersensitivity. AFM experiments showed that wild-type Rap1 and the Rap1[R580A] mutant interact with DNA over 16 successive binding sites, leading to local DNA stiffening but not to accumulation of the observed local distortion. Therefore, Rap1 may cause permanganate hypersensitivity of DNA by forming a pocket between the reactive cytosine and Arg580, driving the permanganate ion towards the C5-C6 bond of the cytosine.

Development of chronic allergic responses by dampening Bcl6-mediated suppressor activity in memory T helper 2 cells

PubMed Central

Ogasawara, Takashi; Hatano, Masahiko; Satake, Hisae; Ikari, Jun; Taniguchi, Toshibumi; Tsuruoka, Nobuhide; Watanabe-Takano, Haruko; Fujimura, Lisa; Sakamoto, Akemi; Hirata, Hirokuni; Sugiyama, Kumiya; Fukushima, Yasutsugu; Nakae, Susumu; Matsumoto, Kenji; Saito, Hirohisa; Fukuda, Takeshi; Kurasawa, Kazuhiro; Tatsumi, Koichiro; Tokuhisa, Takeshi

2017-01-01

Mice deficient in the transcriptional repressor B-cell CLL/lymphoma 6 (Bcl6) exhibit similar T helper 2 (TH2) immune responses as patients with allergic diseases. However, the molecular mechanisms underlying Bcl6-directed regulation of TH2 cytokine genes remain unclear. We identified multiple Bcl6/STAT binding sites (BSs) in TH2 cytokine gene loci. We found that Bcl6 is modestly associated with the BSs, and it had no significant effect on cytokine production in newly differentiated TH2 cells. Contrarily, in memory TH2 (mTH2) cells derived from adaptively transferred TH2 effectors, Bcl6 outcompeted STAT5 for binding to TH2 cytokine gene loci, particularly Interleukin4 (Il4) loci, and attenuated GATA binding protein 3 (GATA3) binding to highly conserved intron enhancer regions in mTH2 cells. Bcl6 suppressed cytokine production epigenetically in mTH2 cells to negatively tune histone acetylation at TH2 cytokine gene loci, including Il4 loci. In addition, IL-33, a pro-TH2 cytokine, diminished Bcl6’s association with loci to which GATA3 recruitment was inversely augmented, resulting in altered IL-4, but not IL-5 and IL-13, production in mTH2 cells but no altered production in newly differentiated TH2 cells. Use of a murine asthma model that generates high levels of pro-TH2 cytokines, such as IL-33, suggested that the suppressive function of Bcl6 in mTH2 cells is abolished in severe asthma. These findings indicate a role of the interaction between TH2-promoting factors and Bcl6 in promoting appropriate IL-4 production in mTH2 cells and suggest that chronic allergic diseases involve the TH2-promoting factor-mediated functional breakdown of Bcl6, resulting in allergy exacerbation. PMID:28096407

Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta 1.

PubMed Central

Liu, C; Adamson, E; Mercola, D

1996-01-01

The early growth response 1 (EGR-1) gene product is a transcription factor with role in differentiation and growth. We have previously shown that expression of exogenous EGR-1 in various human tumor cells unexpectedly and markedly reduces growth and tumorigenicity and, conversely, that suppression of endogenous Egr-1 expression by antisense RNA eliminates protein expression, enhances growth, and promotes phenotypic transformation. However, the mechanism of these effects remained unknown. The promoter of human transforming growth factor beta 1 (TGF-beta 1) contains two GC-rich EGR-1 binding sites. We show that expression of EGR-1 in human HT-1080 fibrosarcoma cells uses increased secretion of biologically active TGF-beta 1 in direct proportion (rPearson = 0.96) to the amount of EGR-1 expressed and addition of recombinant human TGF-beta 1 is strongly growth-suppressive for these cells. Addition of monoclonal anti-TGF-beta 1 antibodies to EGR-1-expressing HT-1080 cells completely reverses the growth inhibitory effects of EGR-1. Reporter constructs bearing the EGR-1 binding segment of the TGF-beta 1 promoter was activated 4- to 6-fold relative to a control reporter in either HT-1080 cells that stably expressed or parental cells cotransfected with an EGR-1 expression vector. Expression of delta EGR-1, a mutant that cannot interact with the corepressors, nerve growth factor-activated factor binding proteins NAB1 and NAB2, due to deletion of the repressor domain, exhibited enhanced transactivation of 2- to 3.5-fold over that of wild-type EGR-1 showing that the reporter construct reflected the appropriate in vivo regulatory context. The EGR-1-stimulated transactivation was inhibited by expression of the Wilms tumor suppressor, a known specific DNA-binding competitor. These results indicate that EGR-1 suppresses growth of human HT-1080 fibrosarcoma cells by induction of TGF-beta 1. Images Fig. 1 Fig. 5 PMID:8876223

Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression.

PubMed

Anandapadamanaban, Madhanagopal; Pilstål, Robert; Andresen, Cecilia; Trewhella, Jill; Moche, Martin; Wallner, Björn; Sunnerhagen, Maria

2016-08-02

MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 Å) presented here is closely similar to wild-type MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members. Copyright © 2016 Elsevier Ltd. All rights reserved.

ME31B globally represses maternal mRNAs by two distinct mechanisms during the Drosophila maternal-to-zygotic transition.

PubMed

Wang, Miranda; Ly, Michael; Lugowski, Andrew; Laver, John D; Lipshitz, Howard D; Smibert, Craig A; Rissland, Olivia S

2017-09-06

In animal embryos, control of development is passed from exclusively maternal gene products to those encoded by the embryonic genome in a process referred to as the maternal-to-zygotic transition (MZT). We show that the RNA-binding protein, ME31B, binds to and represses the expression of thousands of maternal mRNAs during the Drosophila MZT. However, ME31B carries out repression in different ways during different phases of the MZT. Early, it represses translation while, later, its binding leads to mRNA destruction, most likely as a consequence of translational repression in the context of robust mRNA decay. In a process dependent on the PNG kinase, levels of ME31B and its partners, Cup and Trailer Hitch (TRAL), decrease by over 10-fold during the MZT, leading to a change in the composition of mRNA-protein complexes. We propose that ME31B is a global repressor whose regulatory impact changes based on its biological context.

Novobiocin binding to NalD induces the expression of the MexAB-OprM pump in Pseudomonas aeruginosa.

PubMed

Chen, Weizhong; Wang, Dan; Zhou, Wenquan; Sang, Hong; Liu, Xichun; Ge, Zhiyun; Zhang, Jin; Lan, Lefu; Yang, Cai-Guang; Chen, Hao

2016-06-01

NalD was reported to be the secondary repressor of the MexAB-OprM multidrug efflux pump, the major system contributing to intrinsic multidrug resistance in Pseudomonas aeruginosa. Here, we show that novobiocin binds directly to NalD, which leads NalD to dissociate from the DNA promoter, and thus de-represses the expression of the MexAB-OprM pump. In addition, we have solved the crystal structure of NalD at a resolution of 2.90 Å. The structural alignment of NalD to its homologue TtgR reveals that the residues N129 and H167 in NalD are involved in its novobiocin-binding ability. We have confirmed the function of these two amino acids by EMSA and plate assay. The results presented here highlight the importance and diversity of regulatory mechanism in bacterial antibiotic resistance, and provide further insight for novel antimicrobial development. © 2016 John Wiley & Sons Ltd.

Discriminating tastes

PubMed Central

Storz, Gisela

2011-01-01

Hfq-binding small RNAs (sRNAs) are critical regulators that form limited base-pairing interactions with target mRNAs in bacteria. These sRNAs have been linked to diverse environmental responses, yet little is known how Hfq-binding sRNAs participate in the regulatory networks associated with each response. We recently described how the Hfq-binding sRNA Spot 42 in Escherichia coli contributes to catabolite repression, a regulatory phenomenon that allows bacteria to consume some carbon sources over others. Spot 42 base pairs with numerous mRNAs encoding enzymes in central and secondary metabolism, redox balancing, and the uptake and consumption of non-preferred carbon sources. Many of the corresponding genes are transcriptionally activated by the Spot 42-repressor CRP, forming a regulatory circuit called a multi-output feedforward loop. We found that this loop influences both the steady-state levels and dynamics of gene regulation. In this article, we discuss how the CRP-Spot 42 feedforward loop is integrated into encompassing networks and how this loop may benefit enteric bacteria facing uncertain and changing nutrient conditions. PMID:21788732

Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

PubMed

Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

2016-02-01

A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

c-Ski inhibits the TGF-beta signaling pathway through stabilization of inactive Smad complexes on Smad-binding elements.

PubMed

Suzuki, Hiroyuki; Yagi, Ken; Kondo, Miki; Kato, Mitsuyasu; Miyazono, Kohei; Miyazawa, Keiji

2004-06-24

c-Ski inhibits transforming growth factor-beta (TGF-beta) signaling through interaction with Smad proteins. c-Ski represses Smad-mediated transcriptional activation, probably through its action as a transcriptional co-repressor. c-Ski also inhibits TGF-beta-induced downregulation of genes such as c-myc. However, mechanisms for transcriptional regulation of target genes by c-Ski have not been fully determined. In this study, we examined how c-Ski inhibits both TGF-beta-induced transcriptional activation and repression. DNA-affinity precipitation analysis revealed that c-Ski enhances the binding of Smad2 and 4, and to a lesser extent Smad3, to both CAGA and TGF-beta1 inhibitory element probes. A c-Ski mutant, which is unable to interact with Smad4, failed to enhance the binding of Smad complex on these probes and to inhibit the Smad-responsive promoter. These results suggest that stabilization of inactive Smad complexes on DNA is a critical event in c-Ski-mediated inhibition of TGF-beta signaling.

SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes.

PubMed

Mason, Amanda G; Slieker, Roderick C; Balog, Judit; Lemmers, Richard J L F; Wong, Chao-Jen; Yao, Zizhen; Lim, Jong-Won; Filippova, Galina N; Ne, Enrico; Tawil, Rabi; Heijmans, Bas T; Tapscott, Stephen J; van der Maarel, Silvère M

2017-06-06

Facioscapulohumeral muscular dystrophy (FSHD) is in most cases caused by a contraction of the D4Z4 macrosatellite repeat on chromosome 4 (FSHD1) or by mutations in the SMCHD1 or DNMT3B gene (FSHD2). Both situations result in the incomplete epigenetic repression of the D4Z4-encoded retrogene DUX4 in somatic cells, leading to the aberrant expression of DUX4 in the skeletal muscle. In mice, Smchd1 regulates chromatin repression at different loci, having a role in CpG methylation establishment and/or maintenance. To investigate the global effects of harboring heterozygous SMCHD1 mutations on DNA methylation in humans, we combined 450k methylation analysis on mononuclear monocytes from female heterozygous SMCHD1 mutation carriers and unaffected controls with reduced representation bisulfite sequencing (RRBS) on FSHD2 and control myoblast cell lines. Candidate loci were then evaluated for SMCHD1 binding using ChIP-qPCR and expression was evaluated using RT-qPCR. We identified a limited number of clustered autosomal loci with CpG hypomethylation in SMCHD1 mutation carriers: the protocadherin (PCDH) cluster on chromosome 5, the transfer RNA (tRNA) and 5S rRNA clusters on chromosome 1, the HOXB and HOXD clusters on chromosomes 17 and 2, respectively, and the D4Z4 repeats on chromosomes 4 and 10. Furthermore, minor increases in RNA expression were seen in FSHD2 myoblasts for some of the PCDHβ cluster isoforms, tRNA isoforms, and a HOXB isoform in comparison to controls, in addition to the previously reported effects on DUX4 expression. SMCHD1 was bound at DNAseI hypersensitivity sites known to regulate the PCDHβ cluster and at the chromosome 1 tRNA cluster, with decreased binding in SMCHD1 mutation carriers at the PCDHβ cluster sites. Our study is the first to investigate the global methylation effects in humans resulting from heterozygous mutations in SMCHD1. Our results suggest that SMCHD1 acts as a repressor on a limited set of autosomal gene clusters, as an observed reduction in methylation associates with a loss of SMCHD1 binding and increased expression for some of the loci.

Crystal structure of the coat protein from the GA bacteriophage: model of the unassembled dimer.

PubMed Central

Ni, C. Z.; White, C. A.; Mitchell, R. S.; Wickersham, J.; Kodandapani, R.; Peabody, D. S.; Ely, K. R.

1996-01-01

There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L. 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R. Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263). The structures differ in the FG loops and in the first turn of the alpha A helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface. Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F. Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010). This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine. PMID:8976557

Crystal structure of the coat protein from the GA bacteriophage: model of the unassembled dimer.

PubMed

Ni, C Z; White, C A; Mitchell, R S; Wickersham, J; Kodandapani, R; Peabody, D S; Ely, K R

1996-12-01

There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 A resolution (R-factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L. 1993, J Mol Biol 234:620-639) or as an unassembled dimer (Ni Cz, Syed R, Kodandapani R. Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255-263). The structures differ in the FG loops and in the first turn of the alpha A helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA-binding site located in an antiparallel beta-sheet across the dimer interface. Three differences involve residues in the center of this concave site: Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA-binding specificity by GA or MS2 coat protein (Lim F. Spingola M, Peabody DS, 1994, J Biol Chem 269:9006-9010). This sequence difference reflects recognition of the nucleotide at position -5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine.

Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene.

PubMed

Hadjighassem, Mahmoud R; Galaraga, Kimberly; Albert, Paul R

2011-01-01

The serotonin-1A (5-HT1A) receptor functions as a pre-synaptic autoreceptor in serotonin neurons that regulates their activity, and is also widely expressed on non-serotonergic neurons as a post-synaptic heteroreceptor to mediate serotonin action. The 5-HT1A receptor gene is strongly repressed by a dual repressor element (DRE), which is recognized by two proteins: Freud-1/CC2D1A and another unknown protein. Here we identify mouse Freud-2/CC2D1B as the second repressor of the 5-HT1A-DRE. Freud-2 shares 50% amino acid identity with Freud-1, and contains conserved structural domains. Mouse Freud-2 bound specifically to the rat 5-HT1A-DRE adjacent to, and partially overlapping, the Freud-1 binding site. By supershift assay using nuclear extracts from L6 myoblasts, Freud-2-DRE complexes were distinguished from Freud-1-DRE complexes. Freud-2 mRNA and protein were detected throughout mouse brain and peripheral tissues. Freud-2 repressed 5-HT1A promoter-reporter constructs in a DRE-dependent manner in non-neuronal (L6) or 5-HT1A-expressing neuronal (NG108-15, RN46A) cell models. In NG108-15 cells, knockdown of Freud-2 using a specific short-interfering RNA reduced endogenous Freud-2 protein levels and decreased Freud-2 bound to the 5-HT1A-DRE as detected by chromatin immunoprecipitation assay, but increased 5-HT1A promoter activity and 5-HT1A protein levels. Taken together, these data show that Freud-2 is the second component that, with Freud-1, mediates dual repression of the 5-HT1A receptor gene at the DRE. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Msx1 Homeodomain Protein Represses the αGSU and GnRH Receptor Genes During Gonadotrope Development

PubMed Central

Xie, Huimin; Cherrington, Brian D.; Meadows, Jason D.; Witham, Emily A.

2013-01-01

Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LβT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at −114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program. PMID:23371388

Msx1 homeodomain protein represses the αGSU and GnRH receptor genes during gonadotrope development.

PubMed

Xie, Huimin; Cherrington, Brian D; Meadows, Jason D; Witham, Emily A; Mellon, Pamela L

2013-03-01

Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LβT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at -114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program.

CalA, a Cyanobacterial AbrB Protein, Interacts with the Upstream Region of hypC and Acts as a Repressor of Its Transcription in the Cyanobacterium Nostoc sp. Strain PCC 7120▿ †

PubMed Central

Agervald, Åsa; Zhang, Xiaohui; Stensjö, Karin; Devine, Ellenor; Lindblad, Peter

2010-01-01

The filamentous, heterocystous, nitrogen-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain, depending on growth conditions, up to two hydrogenases directly involved in hydrogen metabolism. HypC is one out of at least seven auxiliary gene products required for synthesis of a functional hydrogenase, specifically involved in the maturation of the large subunit. In this study we present a protein, CalA (Alr0946 in the genome), belonging to the transcription regulator family AbrB, which in protein-DNA assays was found to interact with the upstream region of hypC. Transcriptional investigations showed that calA is cotranscribed with the downstream gene alr0947, which encodes a putative protease from the abortive infection superfamily, Abi. CalA was shown to interact specifically not only with the upstream region of hypC but also with its own upstream region, acting as a repressor on hypC. The bidirectional hydrogenase activity was significantly downregulated when CalA was overexpressed, demonstrating a correlation with the transcription factor, either direct or indirect. In silico studies showed that homologues to both CalA and Alr0947 are highly conserved proteins within cyanobacteria with very similar physical organizations of the corresponding structural genes. Possible functions of the cotranscribed downstream protein Alr0947 are presented. In addition, we present a three-dimensional (3D) model of the DNA binding domain of CalA and putative DNA binding mechanisms are discussed. PMID:20023111

Up-regulated transcriptional repressors SnoN and Ski bind Smad proteins to antagonize transforming growth factor-beta signals during liver regeneration.

PubMed

Macias-Silva, Marina; Li, Wei; Leu, Julia I; Crissey, Mary Ann S; Taub, Rebecca

2002-08-09

Transforming growth factor-beta (TGF-beta) functions as an antiproliferative factor for hepatocytes. However, for unexplained reasons, hepatocytes become resistant to TGF-beta signals and can proliferate despite the presence of TGF-beta during liver regeneration. TGF-beta is up-regulated during liver regeneration, although it is not known whether it is active or latent. TGF-beta activity may be examined by assessing Smad activation, a downstream signaling pathway. Smad pathway activation during liver regeneration induced by partial hepatectomy or CC4 injury was examined by assessing the levels of phospho-Smad2 and Smad2-Smad4 complexes. We found that Smad proteins were slightly activated in quiescent liver, but that their activation was further enhanced in regenerating liver. Interestingly, TGF-beta/Smad pathway inhibitors (SnoN and Ski) were up-regulated during regeneration, and notably, SnoN was induced mainly in hepatocytes. SnoN and Ski are transcriptional repressors that may render some cells resistant to TGF-beta via binding Smad proteins. Complexes between SnoN, Ski, and the activated Smad proteins were detected from 2 to 120 h during the major proliferative phase in regenerating liver. Inhibitory complexes decreased after liver mass restitution (5-15 days), suggesting that persistently activated Smad proteins might participate in returning the liver to a quiescent state. Our data show that active TGF-beta/Smad signals are present during regeneration and suggest that SnoN/Ski induction might explain hepatocyte resistance to TGF-beta during the proliferative phase.