Transcriptional Regulatory Sequences Research Articles

Transcriptional regulatory sequence divergence between mouse and bat modifies forelimb length

Transcriptional regulatory sequence divergence between mouse and bat modifies forelimb length

Histone Acetylation and Methylation at Sites Initiating Divergent Polycistronic Transcription in Trypanosoma cruzi

Trypanosomes are ancient eukaryotic parasites in which the protein-coding genes, organized in large polycistronic clusters on both strands, are transcribed from as yet unidentified promoters. In an effort to reveal transcriptional initiation sites, we examined the Trypanosoma cruzi genome for histone modification patterns shown to be linked to active genes in various organisms. Here, we show that acetylated and methylated histones were found to be enriched at strand switch regions of divergent gene arrays, not at convergent clusters or intra- and intergenic regions within clusters. The modified region showed a bimodular profile with two peaks centered over the 5'-regions of the gene pair flanking the strand switch region. This pattern, which demarcates polycistronic transcription units originating from bidirectional initiation sites, is likely to be common in kinetoplastid parasites as well as in other organisms with polycistronic transcription. In contrast, no acetylation was found at promoters of the highly expressed rRNA and spliced leader genes or satellite DNA or at tested retrotransposonal elements. These results reveal, for the first time, the presence of specific epigenetic marks in T. cruzi with potential implications for transcriptional regulation; they indicate that both histone modifications and bidirectional transcription are evolutionarily conserved.

Characterization of the antimicrobial peptide attacin loci from Glossina morsitans.

The antimicrobial peptide Attacin is an immune effector molecule that can inhibit the growth of gram-negative bacteria. In Glossina morsitans morsitans, which serves as the sole vectors of African trypanosomes, Attacins also play a role in trypanosome resistance, and in maintaining parasite numbers at homeostatic levels in infected individuals. We characterized the attacin encoding loci from a Bacterial Artificial Chromosome (BAC) library. The attacin genes are organized into three clusters. Cluster 1 contains two attacin (attA) genes located in head-to-head orientation, cluster 2 contains two closely related genes (attA and attB) located in a similar transcriptional orientation, and cluster 3 contains a single attacin gene (attD). Coding and transcription regulatory sequences of attA and attB are nearly identical, but differ significantly from attD. Putative AttA and AttB have signal peptide sequences, but lack the pro domain typically present in insect Attacins. Putative AttD lacks both domains. Analysis of attacin cDNA sequences shows polymorphisms that could arise either from allelic variations or from the presence of additional attacin genomic loci. Real time-PCR analysis reveals that attA and attB expression is induced in the fat body of flies per os challenged with Escherichia coli and parasitized with trypanosomes. In the midgut, expression of these attacins is similarly induced following microbial challenge, but reduced in response to parasite infections. Transcription of AttD is significantly less relative to the other two genes, and is preferentially induced in the fat body of parasitized flies. These results indicate that the different attacin genes may be differentially regulated.

Spatial Regulation of nanos Is Required for Its Function in Dendrite Morphogenesis

Spatial control of mRNA translation can generate cellular asymmetries and functional specialization of polarized cells like neurons. A requirement for the translational repressor Nanos (Nos) in the Drosophila larval peripheral nervous system (PNS) implicates translational control in dendrite morphogenesis [1]. Nos was first identified by its requirement in the posterior of the early embryo for abdomen formation [2]. Nos synthesis is targeted to the posterior pole of the oocyte and early embryo through translational repression of unlocalized nos mRNA coupled with translational activation of nos mRNA localized at the posterior pole [3, 4]. Abolishment of nos localization prevents abdominal development, whereas translational derepression of unlocalized nos mRNA suppresses head/thorax development, emphasizing the importance of spatial regulation of nos mRNA [3, 5]. Loss and overexpression of Nos affect dendrite branching complexity in class IV dendritic arborization (da) neurons, suggesting that nos also might be regulated in these larval sensory neurons [1]. Here, we show that localization and translational control of nos mRNA are essential for da neuron morphogenesis. RNA-protein interactions that regulate nos translation in the oocyte and early embryo also regulate nos in the PNS. Live imaging of nos mRNA shows that the cis-acting signal responsible for posterior localization in the oocyte/embryo mediates localization to the processes of class IV da neurons but suggests a different transport mechanism. Targeting of nos mRNA to the processes of da neurons may reflect a local requirement for Nos protein in dendritic translational control.

Chance and necessity in chromosomal gene distributions

By analyzing the spacing of genes on chromosomes, we find that transcriptional and RNA-processing regulatory sequences outside coding regions leave footprints on the distribution of intergenic distances. Using analogies between genes on chromosomes and one-dimensional gases, we constructed a statistical null model. We used this to estimate typical upstream and downstream regulatory sequence sizes in various species. Deviations from this model reveal bi-directional transcriptional regulatory regions in Saccharomyces cerevisiae and bi-directional terminators in Escherichia coli.

Identification of a Coronavirus Transcription Enhancer

Coronavirus (CoV) transcription includes a discontinuous mechanism during the synthesis of sub-genome-length minus-strand RNAs leading to a collection of mRNAs in which the 5' terminal leader sequence is fused to contiguous genome sequences. It has been previously shown that transcription-regulating sequences (TRSs) preceding each gene regulate transcription. Base pairing between the leader TRS (TRS-L) and the complement of the body TRS (cTRS-B) in the nascent RNA is a determinant factor during CoV transcription. In fact, in transmissible gastroenteritis CoV, a good correlation has been observed between subgenomic mRNA (sg mRNA) levels and the free energy (DeltaG) of TRS-L and cTRS-B duplex formation. The only exception was sg mRNA N, the most abundant sg mRNA during viral infection in spite of its minimum DeltaG associated with duplex formation. We postulated that additional factors should regulate transcription of sg mRNA N. In this report, we have described a novel transcription regulation mechanism operating in CoV by which a 9-nucleotide (nt) sequence located 449 nt upstream of the N gene TRS core sequence (CS-N) interacts with a complementary sequence just upstream of CS-N, specifically increasing the accumulation of sg mRNA N. Alteration of this complementarity in mutant replicon genomes showed a correlation between the predicted stability of the base pairing between 9-nt sequences and the accumulation of sg mRNA N. This interaction is exclusively conserved in group 1a CoVs, the only CoV subgroup in which the N gene is not the most 3' gene in the viral genome. This is the first time that a long-distance RNA-RNA interaction regulating transcriptional activity specifically enhancing the transcription of one gene has been described to occur in CoVs.

Citations

Citations

Structural and Functional Analyses of the Severe Acute Respiratory Syndrome Coronavirus Endoribonuclease Nsp15

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3′ of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.

The transcription factor encyclopedia, TFe

Event Abstract Back to Event The transcription factor encyclopedia, TFe Wyeth Wasserman1*, Dimas Yusuf1 and Stefanie Butland1 1 University of British Columbia, Canada The willingness of young researchers to participate in online community-curated reference materials such as Wikipedia offers a new opportunity to develop reference resources for biological research. The goal of the TFe is to create an online encyclopedic collection of well-studied transcription factor (TF) proteins, combining a mixture of both hand-curated and automatically-populated content to provide users with a wide set of information relevant to a TF of their interest. Automatically-populated TFe content includes TF binding sites and target genes imported from the PAZAR database of transcription factor and regulatory sequence information (www.pazar.info), DNA-binding domain classification and structure predictions, interactions, phenotypes associated with TF defects, and other external resources. Authors expert in a particular TF contribute abstract-length summary text, figures, a brief reading-list, and additions to or comments on any of the automatically-populated content. As motivation for authors, a static version of the entries at a specific moment in time will be published as brief review articles. The TFe builds upon the MediaWiki software for text editing of individual sections of content into an intuitive interface featuring content-related sections accessible by selection of tabs. The second cohort of authors is currently populating the TFe with articles and providing feedback to direct further refinement of instructional materials and content layout. A focus on neurological transcription factors is planned for the third cohort of authors. Conference: Neuroinformatics 2008, Stockholm, Sweden, 7 Sep - 9 Sep, 2008. Presentation Type: Poster Presentation Topic: Genomics and Genetics Citation: Wasserman W, Yusuf D and Butland S (2008). The transcription factor encyclopedia, TFe. Front. Neuroinform. Conference Abstract: Neuroinformatics 2008. doi: 10.3389/conf.neuro.11.2008.01.079 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Jul 2008; Published Online: 28 Jul 2008. * Correspondence: Wyeth Wasserman, University of British Columbia, Vancouver, Canada, wyeth@cmmt.ubc.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Wyeth Wasserman Dimas Yusuf Stefanie Butland Google Wyeth Wasserman Dimas Yusuf Stefanie Butland Google Scholar Wyeth Wasserman Dimas Yusuf Stefanie Butland PubMed Wyeth Wasserman Dimas Yusuf Stefanie Butland Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems

BackgroundEmploying genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures.ResultsTo enable efficient cloning and manipulation of genomic DNA fragments for the purposes of gene expression and reporter-gene studies we have combined aspects of the Gateway system and a bacteriophage-based homologous recombination (i.e. recombineering) system. To apply the method for characterising plant genes we developed novel Gateway and plant transformation vectors that are of small size and incorporate selectable markers which enable efficient identification of recombinant clones. We demonstrate that the genomic coding region of a gene can be directly cloned into a Gateway Entry vector by recombineering enabling its subsequent transfer to Gateway Expression vectors. We also demonstrate how the coding and regulatory regions of a gene can be directly cloned into a plant transformation vector by recombineering. This construct was then rapidly converted into a novel Gateway Expression vector incorporating cognate 5' and 3' regulatory regions by using recombineering to replace the intervening coding region with the Gateway Destination cassette. Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector. We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression.ConclusionWe describe a novel combination of the favourable attributes of the Gateway and recombineering systems to enable efficient cloning and manipulation of genomic DNA clones for more effective characterisation of gene function. Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.

Finding cis-regulatory elements using comparative genomics: Some lessons from ENCODE data

Identification of functional genomic regions using interspecies comparison will be most effective when the full span of relationships between genomic function and evolutionary constraint are utilized. We find that sets of putative transcriptional regulatory sequences, defined by ENCODE experimental data, have a wide span of evolutionary histories, ranging from stringent constraint shown by deep phylogenetic comparisons to recent selection on lineage-specific elements. This diversity of evolutionary histories can be captured, at least in part, by the suite of available comparative genomics tools, especially after correction for regional differences in the neutral substitution rate. Putative transcriptional regulatory regions show alignability in different clades, and the genes associated with them are enriched for distinct functions. Some of the putative regulatory regions show evidence for recent selection, including a primate-specific, distal promoter that may play a novel role in regulation.

PAP: a comprehensive workbench for mammalian transcriptional regulatory sequence analysis

Given the recent explosion of publications that employ microarray technology to monitor genome-wide expression and that correlate these expression changes to biological processes or to disease states, the determination of the transcriptional regulation of these co-expressed genes is the next major step toward deciphering the genetic network governing the pathway or disease under study. Although computational approaches have been proposed for this purpose, there is no integrated and user-friendly software application that allows experimental biologists to tackle this problem in higher eukaryotes. We have previously reported a systematic, statistical model of mammalian transcriptional regulatory sequence analysis. We have now made crucial extensions to this model and have developed a comprehensive, user-friendly web application suite termed the Promoter Analysis Pipeline (PAP). PAP is available at: http://bioinformatics.wustl.edu/webTools/portalModule/PromoterSearch.do

PAZAR: a framework for collection and dissemination of cis-regulatory sequence annotation

PAZAR is an open-access and open-source database of transcription factor and regulatory sequence annotation with associated web interface and programming tools for data submission and extraction.

Pre-B-Cell Leukemia Transcription Factor 1 Regulates Expression of Valosin-Containing Protein, a Gene Involved in Cancer Growth

Valosin-containing protein (VCP) is involved in a wide variety of cellular functions. Our previous studies showed that the enhanced expression of VCP in cancer cells correlated with invasion and metastasis of cancers. Here, the regulatory mechanism for VCP transcription was investigated. Luciferase reporter constructs containing serially deleted 5'-flanking region of the VCP gene were transfected into MCF7 mammary carcinoma cell line, in which VCP was abundantly expressed. The deletion and mutation at the two binding motifs for pre-B-cell leukemia transcription factor 1 (PBX1) reduced the luciferase activity, indicating that these two PBX1 motifs mediated the transactivation of the VCP gene. Chromatin immunoprecipitation assay showed the binding of PBX-1 to the 5'-flanking region of the VCP gene. The knockdown of PBX1 by siRNA decreased the expression level of VCP. VCP is reported to maintain cell viability after the treatment of tumor necrosis factor-alpha. The viability of tumor necrosis factor-alpha-treated cells was significantly reduced in PBX1 knockdown MCF7. These findings indicate that PBX1 plays a crucial role in VCP expression and function and that the PBX-VCP pathway might be important for cell survival under cytokine stress.

Novel candidate targets of Wnt/β-catenin signaling in hepatoma cells

The activity of β-catenin/TCF, the key component of Wnt signaling pathway, is frequently deregulated in HCC, resulting in the activation of genes whose dysregulation has significant consequences on tumor development. Therefore, identifying the target genes of Wnt signaling is important for understanding β-catenin-mediated carcinogenesis. We analyzed the transcriptome profile of human hepatoma cell lines using cDNA microarrays representing 15,127 unique, liver-enriched gene loci to identify the target genes of β-catenin-mediated transcription ( p < 0.005). This analysis yielded 130 potential Wnt-associated classifier genes, and we found 33 of them contain consensus TCF-binding sites in presumptive transcriptional regulatory sequences. These genes were, then, tested for their Wnt-dependence of expression in experimental models of Wnt activation. Genes such as RPL29, NEDD4L, FUT8, LYZ, STMN2, STARD7 and KIAA0998 were proven to be up-regulated upon Wnt/β-catenin activation. Gene ontology analysis of the 33 candidate genes indicated the presence of functional categories relevant to Wnt pathway such as cell growth, proliferation, adhesion and signal transduction. In conclusion, we identified a number of candidate Wnt/β-catenin target genes that can be useful for studying the role of altered Wnt signaling in liver cancer development, and showed that some of them might be direct targets of Wnt signaling in hepatoma cells.

Comparative Analysis of Twelve Genomes of Three Novel Group 2c and Group 2d Coronaviruses Reveals Unique Group and Subgroup Features

Twelve complete genomes of three novel coronaviruses-bat coronavirus HKU4 (bat-CoV HKU4), bat-CoV HKU5 (putative group 2c), and bat-CoV HKU9 (putative group 2d)-were sequenced. Comparative genome analysis showed that the various open reading frames (ORFs) of the genomes of the three coronaviruses had significantly higher amino acid identities to those of other group 2 coronaviruses than group 1 and 3 coronaviruses. Phylogenetic trees constructed using chymotrypsin-like protease, RNA-dependent RNA polymerase, helicase, spike, and nucleocapsid all showed that the group 2a and 2b and putative group 2c and 2d coronaviruses are more closely related to each other than to group 1 and 3 coronaviruses. Unique genomic features distinguishing between these four subgroups, including the number of papain-like proteases, the presence or absence of hemagglutinin esterase, small ORFs between the membrane and nucleocapsid genes and ORFs (NS7a and NS7b), bulged stem-loop and pseudoknot structures downstream of the nucleocapsid gene, transcription regulatory sequence, and ribosomal recognition signal for the envelope gene, were also observed. This is the first time that NS7a and NS7b downstream of the nucleocapsid gene has been found in a group 2 coronavirus. The high Ka/Ks ratio of NS7a and NS7b in bat-CoV HKU9 implies that these two group 2d-specific genes are under high selective pressure and hence are rapidly evolving. The four subgroups of group 2 coronaviruses probably originated from a common ancestor. Further molecular epidemiological studies on coronaviruses in the bats of other countries, as well as in other animals, and complete genome sequencing will shed more light on coronavirus diversity and their evolutionary histories.

Characterization of White Bream Virus Reveals a Novel Genetic Cluster of Nidoviruses

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

Genome-Wide Analysis of Protein-DNA Interactions

The human genome is predominantly composed of nonprotein-coding sequences whose function remains largely undefined. A significant portion of the noncoding DNA is believed to serve as transcriptional regulatory elements that control gene expression in specific cell types at appropriate developmental stages. Identifying these regulatory sequences and determining the mechanisms by which they act present a great challenge in the postgenomic era. Previous investigations using genetic, molecular, and biochemical approaches have uncovered a large number of proteins involved in regulating transcription. Knowledge of the genomic locations of DNA binding for these proteins in the nucleus should define the identity and nature of the transcriptional regulatory sequences and reveal the gene regulatory networks in cells. Chromatin immunoprecipitation (ChIP) is a common method for detecting interactions between a protein and a DNA sequence in vivo. In recent years, this method has been combined with DNA microarrays and other high-throughput technologies to enable genome-wide identification of DNA-binding sites for various nuclear proteins. Here, we review recent advances in ChIP-based methods for genome-wide detection of protein-DNA interactions, and discuss their significance in enhancing our knowledge of the gene regulatory networks and epigenetic mechanisms in cells.

Putative cis -Acting Stem-Loops in the 5′ Untranslated Region of the Severe Acute Respiratory Syndrome Coronavirus Can Substitute for Their Mouse Hepatitis Virus Counterparts

Consensus covariation-based secondary structural models for the 5' 140 nucleotides of the 5' untranslated regions (5'UTRs) from mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SCoV) were developed and predicted three major helical stem-loop structures, designated stem-loop 1 (SL1), SL2, and SL4. The SCoV 5'UTR was predicted to contain a fourth stem-loop, named SL3, in which the leader transcriptional regulatory sequence (TRS) is folded into a hairpin loop. cDNAs corresponding to MHV/SCoV chimeric genomes were constructed by replacing the complete MHV 5'UTR with the corresponding SCoV sequence and by separately replacing MHV 5'UTR putative SL1, putative SL2, TRS, and putative SL4 with the corresponding SCoV sequences. Chimeric genomes were transcribed in vitro, and viruses were recovered after electroporation into permissive cells. Genomes in which the MHV 5'UTR SL1, SL2, and SL4 were individually replaced by their SCoV counterparts were viable. Chimeras containing the complete SCoV 5'UTR or the predicted SCoV SL3 were not viable. A chimera containing the SCoV 5'UTR in which the SCoV TRS was replaced with the MHV TRS was also not viable. The chimera containing the entire SCoV 5'UTR failed to direct the synthesis of any virus-specific RNA. Replacing the SCoV TRS with the MHV TRS in the MHV/5'UTR SCoV chimera permitted the synthesis of minus-sense genome-sized RNA but did not support the production of positive- or minus-sense subgenomic RNA7. A similar phenotype was obtained with the MHV/SCoV SL3 chimera. These results suggest a role for the TRS in the replication of minus-sense genomic RNA in addition to its known function in subgenomic RNA synthesis.

Detection of variability in apo(a) gene transcription regulatory sequences using the DGGE method

BackgroundIncreased lipoprotein(a), Lp(a), concentration is an independent risk factor for premature atherosclerosis. Apolipoprotein(a), apo(a), determines properties of the lipoprotein and its production rate is the limiting step in Lp(a) particle formation. MethodsSubjects covering the whole range of Lp(a) concentration were separated into quintiles. A randomly chosen sample from each quintile was derived, there being a total number of 713 individuals. The DGGE method was used to scan the known transcription regulatory regions of apo(a) gene (promoter; DHII and DHIII enhancers) for variability and its distribution across quintiles. ResultsBesides 5 previously reported nucleotide substitutions (+121 G>A; +93 C>T; −1712 G>T; −1617 C>A; −1230 A>G) 16 unreported rare sequence variants were detected. All polymorphic variants were distributed throughout the quintiles with several significant differences. The novel +62 C variant was found only among individuals with Lp(a) levels over 16 mg/dl. ConclusionThe apo(a) gene transcription regulatory regions were not revealed to be extremely polymorphic. However, we should consider a combined effect of all polymorphic sites from the whole apo(a) gene locus, including the apo(a) gene length polymorphism, when dealing with high population variability of Lp(a) levels.