Subnuclear localization of the active variant surface glycoprotein gene expression site in Trypanosoma brucei

In Trypanosoma brucei, transcription by RNA polymerase II and 5′ capping of messenger RNA are uncoupled: a capped spliced leader is trans spliced to every RNA. This decoupling makes it possible to have protein-coding gene transcription driven by RNA polymerase I. Indeed, indirect evidence suggests that the genes for the major surface glycoproteins, variant surface glycoproteins (VSGs) in bloodstream-form trypanosomes, are transcribed by RNA polymerase I. In a single trypanosome, only one VSG expression site is maximally transcribed at any one time, and it has been speculated that transcription takes place at a unique site within the nucleus, perhaps in the nucleolus. We tested this by using fluorescence in situ hybridization. With probes that cover about 50 kb of the active 221 expression site, we detected nuclear transcripts of this site in a single fluorescent spot, which did not colocalize with the nucleolus. Analysis of marker gene-tagged active expression site DNA by fluorescent DNA in situ hybridization confirmed the absence of association with the nucleolus. Even an active expression site in which the promoter had been replaced by an rDNA promoter did not colocalize with the nulceolus. As expected, marker genes inserted in the rDNA array predominantly colocalize with the nucleolus, whereas the tubulin gene arrays do not. We conclude that transcription of the active VSG expression site does not take place in the nucleolus.

Profound misregulation of muscle-specific gene expression in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by an insidious onset and progressive course. The disease has a frequency of about 1 in 20,000 and is transmitted in an autosomal dominant fashion with almost complete penetrance. Deletion of an integral number of tandemly arrayed 3.3-kb repeat units (D4Z4) on chromosome 4q35 is associated with FSHD but otherwise the molecular basis of the disease and its pathophysiology remain obscure. Comparison of mRNA populations between appropriate cell types can facilitate identification of genes relevant to a particular biological or pathological process. In this report, we have compared mRNA populations of FSHD and normal muscle. Unexpectedly, the dystrophic muscle displayed profound alterations in gene expression characterized by severe underexpression or overexpression of specific mRNAs. Intriguingly, many of the deregulated mRNAs are muscle specific. Our results suggest that a global misregulation of gene expression is the underlying basis for FSHD, distinguishing it from other forms of muscular dystrophy. The experimental approach used here is applicable to any genetic disorder whose pathogenic mechanism is incompletely understood.

Epidermal muscle attachment site-specific target gene expression and interference with myotube guidance in response to ectopic stripe expression in the developing Drosophila epidermis

The egr-type zinc-finger transcription factor encoded by the Drosophila gene stripe (sr) is expressed in a subset of epidermal cells to which muscles attach during late stages of embryogenesis. We report loss-of-function and gain-of-function experiments indicating that sr activity provides ectodermal cells with properties required for the establishment of a normal muscle pattern during embryogenesis and for the differentiation of tendon-like epidermal muscle attachment sites (EMA). Our results show that sr encodes a transcriptional activator which acts as an autoregulated developmental switch gene. sr activity controls the expression of EMA-specific target genes in cells of ectodermal but not of mesodermal origin. sr-expressing ectodermal cells generate long-range signals that interfere with the spatial orientation of the elongating myotubes.

Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization

Tuberculosis is a chronic infectious disease that is transmitted by cough-propelled droplets that carry the etiologic bacterium, Mycobacterium tuberculosis. Although currently available drugs kill most isolates of M. tuberculosis, strains resistant to each of these have emerged, and multiply resistant strains are increasingly widespread. The growing problem of drug resistance combined with a global incidence of seven million new cases per year underscore the urgent need for new antituberculosis therapies. The recent publication of the complete sequence of the M. tuberculosis genome has made possible, for the first time, a comprehensive genomic approach to the biology of this organism and to the drug discovery process. We used a DNA microarray containing 97% of the ORFs predicted from this sequence to monitor changes in M. tuberculosis gene expression in response to the antituberculous drug isoniazid. Here we show that isoniazid induced several genes that encode proteins physiologically relevant to the drug’s mode of action, including an operonic cluster of five genes encoding type II fatty acid synthase enzymes and fbpC, which encodes trehalose dimycolyl transferase. Other genes, not apparently within directly affected biosynthetic pathways, also were induced. These genes, efpA, fadE23, fadE24, and ahpC, likely mediate processes that are linked to the toxic consequences of the drug. Insights gained from this approach may define new drug targets and suggest new methods for identifying compounds that inhibit those targets.

Directing gene expression to cerebellar granule cells using γ-aminobutyric acid type A receptor α6 subunit transgenes

Expression of the γ-aminobutyric acid type A receptor α6 subunit gene is restricted to differentiated granule cells of the cerebellum and cochlear nucleus. The mechanisms underlying this limited expression are unknown. Here we have characterized the expression of a series of α6-based transgenes in adult mouse brain. A DNA fragment containing a 1-kb portion upstream of the start site(s), together with exons 1–8, can direct high-level cerebellar granule cell-specific reporter gene expression. Thus powerful granule cell-specific determinants reside within the 5′ half of the α6 subunit gene body. This intron-containing transgene appears to lack the cochlear nucleus regulatory elements. It therefore provides a cassette to deliver gene products solely to adult cerebellar granule cells.

A Toolbox for Quantitative Gene Expression in Varroa destructor : RNA Degradation in Field Samples and Systematic Analysis of Reference Gene Stability

Funding: This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 613960 (SMARTBEES) (http://www.smartbees-fp7.eu/) and Veterinary Medicines Directorate, Department for Environment Food & Rural Affairs (Project # VM0517) (https://www.gov.uk/government/organisations/veterinary-medicines-directorate). CHM was supported by a Biosciences Knowledge Transfer Network Biotechnology and Biological Sciences Research Council (KTN-BBSRC CASE) Studentship (BB/L502467/1) (http://www.bbsrc.ac.uk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments We gratefully acknowledge Mr Sebastian Bacz’s expert help and advice with beekeeping.

Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules

FUNDING This work was supported by the Biotechnology and Biological Sciences Research Council [BB/I003746/1 to S.H., BB/M001695/1 to S.H and Y.N]

Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression

Inorganic arsenic, a human carcinogen, is enzymatically methylated for detoxication, consuming S-adenosyl-methionine (SAM) in the process. The fact that DNA methyltransferases (MeTases) require this same methyl donor suggests a role for methylation in arsenic carcinogenesis. Here we test the hypothesis that arsenic-induced initiation results from DNA hypomethylation caused by continuous methyl depletion. The hypothesis was tested by first inducing transformation in a rat liver epithelial cell line by chronic exposure to low levels of arsenic, as confirmed by the development of highly aggressive, malignant tumors after inoculation of cells into Nude mice. Global DNA hypomethylation occurred concurrently with malignant transformation and in the presence of depressed levels of S-adenosyl-methionine. Arsenic-induced DNA hypomethylation was a function of dose and exposure duration, and remained constant even after withdrawal of arsenic. Hyperexpressibility of the MT gene, a gene for which expression is clearly controlled by DNA methylation, was also detected in transformed cells. Acute arsenic or arsenic at nontransforming levels did not induce global hypomethylation of DNA. Whereas transcription of DNA MeTase was elevated, the MeTase enzymatic activity was reduced with arsenic transformation. Taken together, these results indicate arsenic can act as a carcinogen by inducing DNA hypomethylation, which in turn facilitates aberrant gene expression, and they constitute a tenable theory of mechanism in arsenic carcinogenesis.

p53-dependent regulation of MDR1 gene expression causes selective resistance to chemotherapeutic agents

Loss of functional p53 paradoxically results in either increased or decreased resistance to chemotherapeutic drugs. The inconsistent relationship between p53 status and drug sensitivity may reflect p53’s selective regulation of genes important to cytotoxic response of chemotherapeutic agents. We reasoned that the discrepant effects of p53 on chemotherapeutic cytotoxicity is due to p53-dependent regulation of the multidrug resistance gene (MDR1) expression in tumors that normally express MDR1. To test the hypothesis that wild-type p53 regulates the endogenous mdr1 gene we stably introduced a trans-dominant negative (TDN) p53 into rodent H35 hepatoma cells that express P-glycoprotein (Pgp) and have wild-type p53. Levels of Pgp and mdr1a mRNA were markedly elevated in cells expressing TDN p53 and were linked to impaired p53 function (both transactivation and transrepression) in these cells. Enhanced mdr1a gene expression in the TDN p53 cells was not secondary to mdr1 gene amplification and Pgp was functional as demonstrated by the decreased uptake of vinblastine. Cytotoxicity assays revealed that the TDN p53 cell lines were selectively insensitive to Pgp substrates. Sensitivity was restored by the Pgp inhibitor reserpine, demonstrating that only drug retention was the basis for loss of drug sensitivity. Similar findings were evident in human LS180 colon carcinoma cells engineered to overexpress TDN p53. Therefore, the p53 inactivation seen in cancers likely leads to selective resistance to chemotherapeutic agents because of up-regulation of MDR1 expression.

Noncytopathic Sindbis virus RNA vectors for heterologous gene expression

Infection of vertebrate cells with alphaviruses normally leads to prodigious expression of virus-encoded genes and a dramatic inhibition of host protein synthesis. Recombinant Sindbis viruses and replicons have been useful as vectors for high level foreign gene expression, but the cytopathic effects of viral replication have limited their use to transient studies. We recently selected Sindbis replicons capable of persistent, noncytopathic growth in BHK cells and describe here a new generation of Sindbis vectors useful for long-term foreign gene expression based on such replicons. Foreign genes of interest as well as the dominant selectable marker puromycin N-acteyltransferase, which confers resistance to the drug puromycin, were expressed as subgenomic transcripts of noncytopathic replicons or defective-interfering genomes complemented in trans by a replicon. Based on these strategies, we developed vectors that can be initiated via either RNA or DNA transfection and analyzed them for their level and stability of foreign gene expression. Noncytopathic Sindbis vectors express reasonably high levels of protein in nearly every cell. These vectors should prove to be flexible tools for the rapid expression of heterologous genes under conditions in which cellular metabolism is not perturbed, and we illustrate their utility with a number of foreign proteins.

Loss of haloperidol induced gene expression and catalepsy in protein kinase A-deficient mice

The antipsychotic drug, haloperidol, elicits the expression of neurotensin and c-fos mRNA in the dorsal lateral region of the striatum and produces an acute cataleptic response in rodents that correlates with the motor side effects of haloperidol in humans. Mice harboring a targeted disruption of the RIIβ subunit of protein kinase A have a profound deficit in cAMP-stimulated kinase activity in the striatum. When treated with haloperidol, RIIβ mutant mice fail to induce either c-fos or neurotensin mRNA and the acute cataleptic response is blocked. However, both wild-type and mutant mice become cataleptic when neurotensin peptide is directly injected into the lateral ventricle, demonstrating that the kinase deficiency does not interfere with the action of neurotensin but rather its synthesis and release. These results establish a direct role for protein kinase A as a mediator of haloperidol induced gene induction and cataleptic behavior.

Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): The effect of isoniazid on gene expression in Mycobacterium tuberculosis

Understanding the effects of the external environment on bacterial gene expression can provide valuable insights into an array of cellular mechanisms including pathogenesis, drug resistance, and, in the case of Mycobacterium tuberculosis, latency. Because of the absence of poly(A)+ mRNA in prokaryotic organisms, studies of differential gene expression currently must be performed either with large amounts of total RNA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences. We have developed an approach to study differences in bacterial mRNA expression that enables amplification by the PCR of a complex mixture of cDNA sequences in a reproducible manner that obviates the confounding effects of selected highly expressed sequences, e.g., ribosomal RNA. Differential expression using customized amplification libraries (DECAL) uses a library of amplifiable genomic sequences to convert total cellular RNA into an amplified probe for gene expression screens. DECAL can detect 4-fold differences in the mRNA levels of rare sequences and can be performed on as little as 10 ng of total RNA. DECAL was used to investigate the in vitro effect of the antibiotic isoniazid on M. tuberculosis, and three previously uncharacterized isoniazid-induced genes, iniA, iniB, and iniC, were identified. The iniB gene has homology to cell wall proteins, and iniA contains a phosphopantetheine attachment site motif suggestive of an acyl carrier protein. The iniA gene is also induced by the antibiotic ethambutol, an agent that inhibits cell wall biosynthesis by a mechanism that is distinct from isoniazid. The DECAL method offers a powerful new tool for the study of differential gene expression.

Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression

The signal transducer and activator of transcription, STAT5b, has been implicated in signal transduction pathways for a number of cytokines and growth factors, including growth hormone (GH). Pulsatile but not continuous GH exposure activates liver STAT5b by tyrosine phosphorylation, leading to dimerization, nuclear translocation, and transcriptional activation of the STAT, which is proposed to play a key role in regulating the sexual dimorphism of liver gene expression induced by pulsatile plasma GH. We have evaluated the importance of STAT5b for the physiological effects of GH pulses using a mouse gene knockout model. STAT5b gene disruption led to a major loss of multiple, sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression were decreased to wild-type female levels in STAT5b−/− males, while female-predominant liver gene products were increased to a level intermediate between wild-type male and female levels. Although these responses are similar to those observed in GH-deficient Little mice, STAT5b−/− mice are not GH-deficient, suggesting that they may be GH pulse-resistant. Indeed, the dwarfism, elevated plasma GH, low plasma insulin-like growth factor I, and development of obesity seen in STAT5b−/− mice are all characteristics of Laron-type dwarfism, a human GH-resistance disease generally associated with a defective GH receptor. The requirement of STAT5b to maintain sexual dimorphism of body growth rates and liver gene expression suggests that STAT5b may be the major, if not the sole, STAT protein that mediates the sexually dimorphic effects of GH pulses in liver and perhaps other target tissues. STAT5b thus has unique physiological functions for which, surprisingly, the highly homologous STAT5a is unable to substitute.

Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type1a receptor (AT1aR)-dependent mechanism. To understand how the level of AT1aRs is altered in this pathological state, we studied the expression of an injected AT1aR promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the α-skeletal actin promoter 1.7-fold whereas the α-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT1aR promoter by 1.6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT1aR promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT1aR promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.

Inhibition of gene expression in human cells through small molecule-RNA interactions

Small molecules that bind their biological receptors with high affinity and selectivity can be isolated from randomized pools of combinatorial libraries. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of HIV-1 gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5′ end of all nascent HIV-1 transcripts. Here we demonstrate the isolation of small TAR RNA-binding molecules from an encoded combinatorial library. We have made an encoded combinatorial tripeptide library of 24,389 possible members from d-and l-alpha amino acids on TentaGel resin. Using on-bead screening we have identified a small family of mostly heterochiral tripeptides capable of structure-specific binding to the bulge loop of TAR RNA. In vitro binding studies reveal stereospecific discrimination when the best tripeptide ligand is compared with diastereomeric peptide sequences. In addition, the most strongly binding tripeptide was shown to suppress transcriptional activation by Tat protein in human cells with an IC50 of ≈50 nM. Our results indicate that tripeptide RNA ligands are cell permeable, nontoxic to cells, and capable of inhibiting expression of specific genes by interfering with RNA-protein interactions.