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PubMed2006

A CCAAT/enhancer-binding protein site at -87 is required for the activation of a novel murine melanocortin 2-receptor promoter at late stages during adipogenesis.

The peptide hormone ACTH stimulates lipolysis and suppresses leptin production in adipocytes via the G protein-coupled receptor, melanocortin 2 receptor (MC2-R). We have shown previously that peroxisome proliferator-activated receptor-gamma2 is the primary factor responsible for transactivation of the already identified murine MC2-R promoter in the differentiating 3T3-L1 adipocyte cell line. In this study we show that despite the activity of this promoter being transient during differentiation, MC2-R message remains elevated at later time points during adipogenesis. Analysis of the late transcripts reveals that they initiate from a transcriptional start site in the first intron of the murine MC2-R. The genomic sequence upstream of this start site acts as an adipocyte-specific promoter whose activation is delayed in differentiation, compared with the upstream promoter. A CCAAT/enhancer-binding protein binding site, 87 bp upstream of the transcriptional initiation site, is necessary for the activity of this promoter, and protein binding analyses reveal that this site is bound by CCAAT/enhancer-binding protein factors. Real-time PCR analysis of mRNA initiating from the two start sites shows that there is a switch in promoter usage from the 5' to the 3' promoter around d 5, indicating the complex regulation of the murine MC2-R during adipogenesis.

3T3-L1 Cells↗

PubMed2006

The structural basis for promoter -35 element recognition by the group IV sigma factors.

The control of bacterial transcription initiation depends on a primary sigma factor for housekeeping functions, as well as alternative sigma factors that control regulons in response to environmental stresses. The largest and most diverse subgroup of alternative sigma factors, the group IV extracytoplasmic function sigma factors, directs the transcription of genes that regulate a wide variety of responses, including envelope stress and pathogenesis. We determined the 2.3-A resolution crystal structure of the -35 element recognition domain of a group IV sigma factor, Escherichia coli sigma(E)4, bound to its consensus -35 element, GGAACTT. Despite similar function and secondary structure, the primary and group IV sigma factors recognize their -35 elements using distinct mechanisms. Conserved sequence elements of the sigma(E) -35 element induce a DNA geometry characteristic of AA/TT-tract DNA, including a rigid, straight double-helical axis and a narrow minor groove. For this reason, the highly conserved AA in the middle of the GGAACTT motif is essential for -35 element recognition by sigma(E)4, despite the absence of direct protein-DNA interactions with these DNA bases. These principles of sigma(E)4/-35 element recognition can be applied to a wide range of other group IV sigma factors.

Amino Acid Sequence↗

PubMed2006

Activation of the chicken Ig-beta locus by the collaboration of scattered regulatory regions through changes in chromatin structure.

A total of 10 B-lymphocyte-specific DNase I hypersensitive sites located in the chicken Ig-beta locus were divided into four regions and combinations of deletions of these regions were carried out. A decrease in transcription of the Ig-beta gene to <3% was demonstrated in cells with deletions in all four regions. The Ig-beta chromatin was resistant to DNase I digestion in these cells. Thus, the collaboration is shown to convert the Ig-beta chromatin from the condensed state to a relaxed state. H3 and H4 acetylation decreased to <8% but H3K4 hypermethylation was observed at the Ig-beta promoter and exon 3. The collaboration of four regions had virtually no effect on CG hypomethylation in the region upstream the transcriptional start site. Accordingly, neither the DNase I general sensitive state in the Ig-beta chromatin nor hyperacetylation of H3 and H4 histones in the promoter proximal region causes H3K4 di-methylation or CG hypomethylation in the promoter. From these analyses, a chromatin situation was found in which both an active state, such as enhanced H3K4 methylation, or CG hypomethylation, and an inactive state, such as DNase I resistance in the Ig-beta chromatin or hypoacetylation of H3 and H4 histones in the Ig-beta locus, coexist.

Acetylation↗

PubMed2006

A mutant thyroid hormone receptor alpha antagonizes peroxisome proliferator-activated receptor alpha signaling in vivo and impairs fatty acid oxidation.

Thyroid hormone regulates the balance between lipolysis and lipogenesis. We previously reported that male mice with a dominant-negative P398H mutation introduced into the TRalpha gene have visceral obesity, hyperleptinemia, and reduced catecholamine-stimulated lipolysis in white adipose tissue. Based on our observation of hepatic steatosis in the TRalpha P398H male mice, we used in vitro and in vivo models to investigate the influence of the TRalpha P398H mutant on peroxisome proliferator-activated receptor-alpha (PPARalpha) signaling. Wild-type TRalpha and the P398H mutant significantly reduced PPARalpha-mediated transcription in transient transfection assays. T(3) reversed the inhibition of PPARalpha action by wild-type TRalpha but not the P398H mutant. Chromatin immunoprecipitation assays demonstrated that the P398H mutant reduces PPARalpha binding to peroxisome proliferator receptor elements. In gel shift assays, the P398H mutant directly bound the peroxisome proliferator-activated receptor response element and inhibited PPARalpha binding, which was not reversed by addition of retinoid X receptor. The TRalpha R384C and PV dominant-negative mutants are not associated in vivo with a metabolic phenotype and had reduced (PV) or absent (R384C) PPARalpha inhibition compared with P398H. The metabolic phenotype of the P398H mutant mice is due, in part, to unique properties of the P398H mutant receptor interfering with PPARalpha signaling. The P398H mutant is a potential probe to characterize the physiological role of thyroid hormone receptor/PPARalpha interactions.

Animals↗

PubMed2005

Isolation and characterization of a lipid transfer protein expressed in ripening fruit of Capsicum chinense.

A novel LTP (CcLTP) from a Capsicum chinense cv Habanero was isolated from a fruit-specific SSH library. While this gene shares similarity with other LTPs, it is considerably larger than any lipid transfer protein reported to date and has a neutral predicted pI. CcLTP is consistently expressed in seedlings from three Capsicum species. It is also present at very high levels in ripening and mature fruit in C. chinense, but not in fruit of any C. annuum or C. frutescens varieties examined. We have obtained 3.8 kb of sequence containing the CcLTP gene and isolated two forms of mRNA transcripts which result from an alternative splicing event. Both transcripts are full-length cDNAs with putative open reading frames of 492 bp and 519 bp, encoding proteins of 164 and 173 amino acids, respectively, which differ only by an insertion of 9 amino acids. Both splice variants are detected consistently via RT-PCR. A 19 bp deletion in the promoter region differentiates C. chinense CcLTP from that of C. annuum and C. frutescens. The protein and its expression are characterized in C. chinense fruit, and a possible role in pepper fruit ripening and maturation is discussed.

Alternative Splicing↗

PubMed2006

Tractor_DB (version 2.0): a database of regulatory interactions in gamma-proteobacterial genomes.

The version 2.0 of Tractor_DB is now accessible at its three international mirrors: www.bioinfo.cu/Tractor_DB, www.tractor.lncc.br and http://www.ccg.unam.mx/tractorDB. This database contains a collection of computationally predicted Transcription Factors' binding sites in gamma-proteobacterial genomes. These data should aid researchers in the design of microarray experiments and the interpretation of their results. They should also facilitate studies of Comparative Genomics of the regulatory networks of this group of organisms. In this paper we describe the main improvements incorporated to the database in the past year and a half which include incorporating information on the regulatory networks of 13-increasing to 30-new gamma-proteobacteria and developing a new computational strategy to complement the putative sites identified by the original weight matrix-based approach. We have also added dynamically generated navigation tabs to the navigation interfaces. Moreover, we developed a new interface that allows users to directly retrieve information on the conservation of regulatory interactions in the 30 genomes included in the database by navigating a map that represents a core of the known Escherichia coli regulatory network.

Binding Sites↗

PubMed2006

Differential dependence of StpA on H-NS in autoregulation of stpA and in regulation of bgl.

StpA has functional similarity to its homologue, the nucleoid structuring protein H-NS. It binds to AT-rich, planar, bent DNA and constrains DNA supercoils. In addition, StpA acts as an RNA chaperone. StpA and H-NS also form heterodimers. However, cellular levels of StpA are low due to repression of stpA by H-NS and negative autoregulation. Here we show that effective (30-fold) repression of stpA transcription requires a downstream regulator element located within the stpA coding region. In addition, we show that StpA represses stpA threefold in an hns null mutant. In contrast, repression of the bgl operon, another H-NS-repressed system, is not achieved by StpA alone. It becomes StpA dependent in the presence of a fusion protein encompassing the N-terminal 37 amino acids of H-NS, which comprise the core of the dimerization domain. StpA also effectively complements H-NS-I119T, a mutant defective in specific DNA binding, in repression of the bgl operon. Thus, StpA complements H-NS proteins defective in DNA binding to repress bgl, while in autoregulation of stpA it acts autonomously, indicating a difference in the mechanisms of repression.

Amino Acid Sequence↗

PubMed2007

Glutamate activates c-fos in glial cells via a novel mechanism involving the glutamate receptor subtype mGlu5 and the transcriptional repressor DREAM.

Activation of c-fos in brain is related to coupling of neuronal activity to gene expression, but also to pathological conditions such as seizures or excitotoxicity-induced cell death. Glutamate activates c-fos in neurons through the calcium-dependent phosphorylation of CREB by ERK and/or CaMKIV kinase pathways downstream NMDA-receptors. In glial cells, however, the activation of c-fos by glutamate is poorly understood. Because glial cells actively modulate neuronal excitability and the brain's response to injury, we studied the mechanisms by which glutamate activates c-fos in rat cortical glial cells. Glutamate potently induced c-fos mRNA in a calcium-dependent manner, as demonstrated by using the calcium chelator BAPTA-AM. Glutamate-induced c-fos mRNA expression was not sensitive to inhibitors of ERK, p38(MAPK), or CaMK pathways, indicating that glial c-fos is activated by a distinct mechanism. Thapsigargin abolished the glutamate effect on c-fos mRNA, indicating ER calcium mobilization. Additionally, glutamate induction of c-fos mRNA was sensitive to the mGluR5 antagonist MPEP but not the NMDA-R antagonist MK-801. In luciferase reporter assays, DRE, which actively represses c-fos by binding the calcium-binding transcriptional repressor DREAM, was activated by glutamate, whereas SRE and CRE were not. Finally, glutamate caused the nuclear export of DREAM in astrocytes, and transfection of astrocytes with a mutant variant of DREAM that constitutively binds DNA inhibited glutamate-induced c-Fos expression. These findings are in sharp contrast to the mechanism described in neurons and suggest a novel pathway activated by glutamate in glial cells that employs mGluR5, ER calcium, and the derepression of c-fos at the DRE.

Animals↗

PubMed2006

Drosophila Reptin and other TIP60 complex components promote generation of silent chromatin.

Histone acetyltransferase (HAT) complexes have been linked to activation of transcription. Reptin is a subunit of different chromatin-remodeling complexes, including the TIP60 HAT complex. In Drosophila, Reptin also copurifies with the Polycomb group (PcG) complex PRC1, which maintains genes in a transcriptionally silent state. We demonstrate genetic interactions between reptin mutant flies and PcG mutants, resulting in misexpression of the homeotic gene Scr. Genetic interactions are not restricted to PRC1 components, but are also observed with another PcG gene. In reptin homozygous mutant cells, a Polycomb response-element-linked reporter gene is derepressed, whereas endogenous homeotic gene expression is not. Furthermore, reptin mutants suppress position-effect variegation (PEV), a phenomenon resulting from spreading of heterochromatin. These features are shared with three other components of TIP60 complexes, namely Enhancer of Polycomb, Domino, and dMRG15. We conclude that Drosophila Reptin participates in epigenetic processes leading to a repressive chromatin state as part of the fly TIP60 HAT complex rather than through the PRC1 complex. This shows that the TIP60 complex can promote the generation of silent chromatin.

Animals↗

PubMed1993

The KROX-20 transcription factor in the rat central and peripheral nervous systems: novel expression pattern of an immediate early gene-encoded protein.

The KROX-20 protein (also termed EGR-2) is encoded by an immediate early gene cloned by cross-hybridization to the Drosophila melanogaster Krüppel gene. It belongs to a class of transcription factors with zinc finger motifs and binding activity to a transcriptional regulatory DNA element termed the early growth response consensus sequence. In the present study the temporospatial expression of KROX-20 was investigated in the central and peripheral nervous systems of normal rats and after various stimuli known to induce immediate early genes, including epileptic seizures, axotomy, pharmacological treatment with glutamate and alpha-adrenergic receptor antagonists, and peripheral noxious stimulation. Immunocytochemistry was performed with a specific polyclonal antiserum generated against a fusion protein containing KROX-20 sequences. In the central nervous system, KROX-20 protein demonstrated distinct constitutive nuclear expression in specific neuronal subpopulations of the cortex, septum, amygdala, olfactory bulb and hypothalamus. In addition, distinct cytoplasmic immunoreactivity was present in spinal and medullary motoneurons, dorsal root ganglion neurons and a few neuronal cell populations of midbrain and forebrain. In the CNS, KROX-20 was only induced by bicuculline-induced epileptic seizures. Topographically, the postictal increase of KROX-20 levels was restricted to areas with constitutive expression, such as cerebral cortex, fornix and amygdala. Induction of KROX-20 peaked at 4-8 h after onset of seizure activity. No increase in immunoreactivity was observed in the hippocampus, the brain region most severely affected by bicuculline-induced seizures. Transection of central and peripheral nerve fibers did not result in KROX-20 induction in axotomized neurons. However, KROX-20 was induced in Schwann-like cells after transection of the sciatic nerve. In contrast to KROX-20, KROX-24, a related transcription factor of the zinc finger family, was markedly induced in hippocampal and spinal neurons following seizures and peripheral noxious stimulation, respectively, as well as in CNS neurons following axotomy. Our data indicate that KROX-20 represents an immediately early gene product with basal expression in selected neuronal populations of the nervous system and a restricted inducibility after intentional stimuli.

Animals↗

PubMed2003

Computational detection of cis -regulatory modules.

MOTIVATION: The transcriptional regulation of a metazoan gene depends on the cooperative action of multiple transcription factors that bind to cis-regulatory modules (CRMs) located in the neighborhood of the gene. By integrating multiple signals, CRMs confer an organism specific spatial and temporal rate of transcription. RESULTS: Based on the hypothesis that genes that are needed in exactly the same conditions might share similar regulatory switches, we have developed a novel methodology to find CRMs in a set of coexpressed or coregulated genes. The ModuleSearcher algorithm finds for a given gene set the best scoring combination of transcription factor binding sites within a sequence window using an A(*)procedure for tree searching. To keep the level of noise low, we use DNA sequences that are most likely to contain functional cis-regulatory information, namely conserved regions between human and mouse orthologous genes. The ModuleScanner performs genomic searches with a predicted CRM or with a user-defined CRM known from the literature to find possible target genes. The validity of a set of putative targets is checked using Gene Ontology annotations. We demonstrate the use and effectiveness of the ModuleSearcher and ModuleScanner algorithms and test their specificity and sensitivity on semi-artificial data. Next, we search for a module in a cluster of gene expression profiles of human cell cycle genes. AVAILABILITY: The ModuleSearcher is available as a web service within the TOUCAN workbench for regulatory sequence analysis, which can be downloaded from http://www.esat.kuleuven.ac.be/~dna/BioI.

Algorithms↗

PubMed2006

Preliminary structural studies of the transcriptional regulator CmeR from Campylobacter jejuni.

In Campylobacter jejuni, a gram-negative bacterial pathogen causing gastroenteritis in humans, the CmeR regulatory protein controls transcription of the multidrug transporter gene operon cmeABC. CmeR belongs to the TetR family of transcriptional regulators. The 210-residue CmeR consists of two functional motifs: an N-terminal DNA-binding domain and a C-terminal ligand-binding domain. It is predicted that the DNA-binding domain interacts directly with target promoters, while the C-terminal motif interacts with inducing ligands (such as bile salts). As an initial step towards confirming this structural model, recombinant CmeR protein containing a 6 x His tag at the N-terminus was crystallized. Crystals of ligand-free CmeR belonged to space group P2(1)2(1)2, with unit-cell parameters a = 37.4, b = 57.6, c = 93.3 A. Diffraction was observed to at least 2.2 A at 100 K. Analysis of the detailed CmeR structure is currently in progress.

Campylobacter jejuni↗

PubMed2006

Mutation and evolutionary analyses identify NR2E1-candidate-regulatory mutations in humans with severe cortical malformations.

Nuclear receptor 2E1 (NR2E1) is expressed in human fetal and adult brains; however, its role in human brain-behavior development is unknown. Previously, we have corrected the cortical hypoplasia and behavioral abnormalities in Nr2e1(-/-) mice using a genomic clone spanning human NR2E1, which bolsters the hypothesis that NR2E1 may similarly play a role in human cortical and behavioral development. To test the hypothesis that humans with abnormal brain-behavior development may have null or hypomorphic NR2E1 mutations, we undertook the first candidate mutation screen of NR2E1 by sequencing its entire coding region, untranslated, splice site, proximal promoter and evolutionarily conserved non-coding regions in 56 unrelated patients with cortical disorders, namely microcephaly. We then genotyped the candidate mutations in 325 unrelated control subjects and 15 relatives. We did not detect any coding region changes in NR2E1; however, we identified seven novel candidate regulatory mutations that were absent from control subjects. We used in silico tools to predict the effects of these candidate mutations on neural transcription factor binding sites (TFBS). Four candidate mutations were predicted to alter TFBS. To facilitate the present and future studies of NR2E1, we also elucidated its molecular evolution, genetic diversity, haplotype structure and linkage disequilibrium by sequencing an additional 94 unaffected humans representing Africa, the Americas, Asia, Europe, the Middle East and Oceania, as well as great apes and monkeys. We detected strong purifying selection, low genetic diversity, 21 novel polymorphisms and five common haplotypes at NR2E1. We conclude that protein-coding changes in NR2E1 do not contribute to cortical and behavioral abnormalities in the patients examined here, but that regulatory mutations may play a role.

Animals↗

PubMed2006

A graph-based motif detection algorithm models complex nucleotide dependencies in transcription factor binding sites.

Given a set of known binding sites for a specific transcription factor, it is possible to build a model of the transcription factor binding site, usually called a motif model, and use this model to search for other sites that bind the same transcription factor. Typically, this search is performed using a position-specific scoring matrix (PSSM), also known as a position weight matrix. In this paper we analyze a set of eukaryotic transcription factor binding sites and show that there is extensive clustering of similar k-mers in eukaryotic motifs, owing to both functional and evolutionary constraints. The apparent limitations of probabilistic models in representing complex nucleotide dependencies lead us to a graph-based representation of motifs. When deciding whether a candidate k-mer is part of a motif or not, we base our decision not on how well the k-mer conforms to a model of the motif as a whole, but how similar it is to specific, known k-mers in the motif. We elucidate the reasons why we expect graph-based methods to perform well on motif data. Our MotifScan algorithm shows greatly improved performance over the prevalent PSSM-based method for the detection of eukaryotic motifs.

Algorithms↗

PubMed2006

In vitro evidence that upstream of N-ras participates in the regulation of parathyroid hormone messenger ribonucleic acid stability.

Calcium and phosphate regulate PTH gene expression posttranscriptionally through the binding of trans-acting factors to a defined cis-acting instability element in the PTH mRNA 3'-untranslated region (UTR). We have previously defined AU-rich binding factor 1 as a PTH mRNA binding and stabilizing protein. We have now identified, by affinity chromatography, Upstream of N-ras (Unr) as another PTH mRNA 3'-UTR binding protein. Recombinant Unr bound the PTH 3'-UTR transcript, and supershift experiments with antibodies to Unr showed that Unr is part of the parathyroid RNA binding complex. Finally, because there is no parathyroid cell line, the functionality of Unr in regulating PTH mRNA levels was demonstrated in cotransfection experiments in heterologous human embryonic kidney 293 cells. Depletion of Unr by small interfering RNA decreased simian virus 40-driven PTH gene expression in human embryonic kidney 293 cells transiently cotransfected with the human PTH gene. Overexpression of Unr increased the rat full-length PTH mRNA levels but not a PTH mRNA lacking the terminal 60-nucleotide cis-acting protein binding region. Unr also stabilized a chimeric GH reporter mRNA that contained the rat PTH 63-nucleotide cis-acting element but not a truncated PTH element. Therefore, Unr binds to the PTH cis element and increases PTH mRNA levels, as does AU-rich binding factor 1. Our results suggest that Unr, together with the other proteins in the RNA binding complex, determines PTH mRNA stability.

3' Untranslated Regions↗

PubMed2006

AnCrpA, a cAMP receptor protein, regulates nif-related gene expression in the cyanobacterium Anabaena sp. strain PCC 7120 grown with nitrate.

Target genes for a cAMP receptor protein, AnCrpA, were screened using an Anabaena oligonucleotide microarray and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Several gene expressions, including some involved in nitrogen fixation, were downregulated in the ancrpA disruptant when cells were grown with nitrate. Electrophoretic mobility shift assays (EMSAs) revealed that AnCrpA bound to the 5' upstream region of nifB, all1439, hesA, all5347, hglE and coxBII in the presence of cAMP, and all of them are related with nitrogen fixation. A possible AnCrpA-binding site in the 5' upstream region of nifB was predicted using hidden Markov model (HMM) software based on the result of in vitro selection of AnCrpA-binding sequences, and the binding was confirmed by EMSA. Thus, AnCrpA regulates the expressions of gene clusters related to nitrogen fixation in the presence of nitrate.

Anabaena↗

PubMed2005

Small-molecule interaction with a five-guanine-tract G-quadruplex structure from the human MYC promoter.

It has been widely accepted that DNA can adopt other biologically relevant structures beside the Watson-Crick double helix. One recent important example is the guanine-quadruplex (G-quadruplex) structure formed by guanine tracts found in the MYC (or c-myc) promoter region, which regulates the transcription of the MYC oncogene. Stabilization of this G-quadruplex by ligands, such as the cationic porphyrin TMPyP4, decreases the transcriptional level of MYC. Here, we report the first structure of a DNA fragment containing five guanine tracts from this region. An unusual G-quadruplex fold, which was derived from NMR restraints using unambiguous model-independent resonance assignment approaches, involves a core of three stacked guanine tetrads formed by four parallel guanine tracts with all anti guanines and a snapback 3'-end syn guanine. We have determined the structure of the complex formed between this G-quadruplex and TMPyP4. This structural information, combined with details of small-molecule interaction, provides a platform for the design of anticancer drugs targeting multi-guanine-tract sequences that are found in the MYC and other oncogenic promoters, as well as in telomeres.

Biomarkers, Tumor↗

PubMed2005

Whole-genome analyses of speciation events in pathogenic Brucellae.

Despite their high DNA identity and a proposal to group classical Brucella species as biovars of Brucella melitensis, the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters, as well as by a marked host range (e.g., Brucella suis for swine, B. melitensis for sheep and goats, and Brucella abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucella species and to B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions, and insertions supports previous indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis. With the exception of a dozen genes, the genetic complements of both B. abortus strains are identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggests that these inactivations may play an important role in the establishment of host specificity and may have been a primary driver of speciation in the genus Brucella. Despite being nonmotile, the brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures and may contribute to differences in host specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g., glycogen, biotin, NAD, and choline) are consistent with adaptation of brucellae to an intracellular life-style.

Bacterial Proteins↗