Molecular cloning of a novel human I-mfa domain-containing protein that differently regulates human T-cell leukemia virus type I and HIV-1 expression.

Regulation of viral genome expression is the result of complex cooperation between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the basic helix-loop-helix repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40, respectively. We show that the HIC protein isoforms present different subcellular localizations, p32 being mainly distributed throughout the cytoplasm, whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T-cells the expression of a luciferase reporter gene driven by the human T-cell leukemia virus type I-long terminal repeat in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Finally, we also show that HIC is able to down-regulate the luciferase expression from the human immunodeficiency virus type 1-long terminal repeat induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of a new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain.

Regulatable and modulable background expression control in prokaryotic synthetic circuits by auxiliary repressor binding sites.

Expression control in synthetic genetic circuitry, for example, for construction of sensitive biosensors, is hampered by the lack of DNA parts that maintain ultralow background yet achieve high output upon signal integration by the cells. Here, we demonstrate how placement of auxiliary transcription factor binding sites within a regulatable promoter context can yield an important gain in signal-to-noise output ratios from prokaryotic biosensor circuits. As a proof of principle, we use the arsenite-responsive ArsR repressor protein from Escherichia coli and its cognate operator. Additional ArsR operators placed downstream of its target promoter can act as a transcription roadblock in a distance-dependent manner and reduce background expression of downstream-placed reporter genes. We show that the transcription roadblock functions both in cognate and heterologous promoter contexts. Secondary ArsR operators placed upstream of their promoter can also improve signal-to-noise output while maintaining effector dependency. Importantly, background control can be released through the addition of micromolar concentrations of arsenite. The ArsR-operator system thus provides a flexible system for additional gene expression control, which, given the extreme sensitivity to micrograms per liter effector concentrations, could be applicable in more general contexts.

Comparative study and identification of potent eukaryotic transcriptional repressors in gene switch systems.

In mammalian cells, proper gene regulation is achieved by the complex interplay of transcription factors that activate or repress gene expression by binding to the regulatory regions of target promoters. While transcriptional activators have been extensively characterised and classified into functional groups, relatively little is known about the comparative strength and cell type-specificity of transcriptional repressors. Here, we have compared the ability of a series of eukaryotic repression domains to silence basal and activated transcription. A series of the most potent repression domains was further tested in the context of a gene therapy gene-switch system in various cell types. The results indicate that the analysed repression domains exert varying silencing activities in different promoter contexts. Furthermore, their potential for gene silencing varies also depending on the cellular context. When multimerised within one chimeric repressor protein, particular combinations of repressor domains were found to display synergistic repressing effects and efficient repression in a panel of cell lines. This approach thus allowed the identification of transcriptional repressors that are both potent and versatile in terms of cellular specificity as a basis for gene switch systems.

Inducible gene and shRNA expression in resident hematopoietic stem cells in vivo.

Hematopoietic stem cells (HSC) are probably the best understood somatic stem cells and often serve as a paradigm for other stem cells. Nevertheless, most current techniques to genetically manipulate them in vivo are either constitutive and/or induced in settings of hematopoietic stress such as after irradiation. Here, we present a conditional expression system that allows for externally controllable transgenesis and knockdown in resident HSCs, based on a lentiviral vector containing a tet-O sequence and a transgenic mouse line expressing a doxycyclin-regulated tTR-KRAB repressor protein. HSCs harvested from tTR-KRAB mice are transduced with the lentiviral vector containing a cDNA (i.e., Green Fluorescent Protein (GFP)) and/or shRNA (i.e., p53) of interest and then transplanted into lethally irradiated recipients. While the vector is effectively repressed by tTR-KRAB during homing and engraftment, robust GFP/shp53 expression is induced on doxycyclin treatment in HSCs and their progeny. Doxycylin-controllable transcription is maintained on serial transplantation, indicating that repopulating HSCs are stably modified by this approach. In summary, this easy to implement conditional system provides inducible and reversible overexpression or knock down of genes in resident HSCs in vivo using a drug devoid of toxic or activating effects.

A glycine-arginine domain in control of the human MRE11 DNA repair protein.

Human MRE11 is a key enzyme in DNA double-strand break repair and genome stability. Human MRE11 bears a glycine-arginine-rich (GAR) motif that is conserved among multicellular eukaryotic species. We investigated how this motif influences MRE11 function. Human MRE11 alone or a complex of MRE11, RAD50, and NBS1 (MRN) was methylated in insect cells, suggesting that this modification is conserved during evolution. We demonstrate that PRMT1 interacts with MRE11 but not with the MRN complex, suggesting that MRE11 arginine methylation occurs prior to the binding of NBS1 and RAD50. Moreover, the first six methylated arginines are essential for the regulation of MRE11 DNA binding and nuclease activity. The inhibition of arginine methylation leads to a reduction in MRE11 and RAD51 focus formation on a unique double-strand break in vivo. Furthermore, the MRE11-methylated GAR domain is sufficient for its targeting to DNA damage foci and colocalization with gamma-H2AX. These studies highlight an important role for the GAR domain in regulating MRE11 function at the biochemical and cellular levels during DNA double-strand break repair.

SOCS-1 protein prevents Janus Kinase/STAT-dependent inhibition of beta cell insulin gene transcription and secretion in response to interferon-gamma.

In the pathogenesis of type I diabetes mellitus, activated leukocytes infiltrate pancreatic islets and induce beta cell dysfunction and destruction. Interferon (IFN)-gamma, tumor necrosis factor-alpha and interleukin (IL)-1 beta play important, although not completely defined, roles in these mechanisms. Here, using the highly differentiated beta Tc-Tet insulin-secreting cell line, we showed that IFN-gamma dose- and time-dependently suppressed insulin synthesis and glucose-stimulated secretion. As described previously IFN-gamma, in combination with IL-1 beta, also induces inducible NO synthase expression and apoptosis (Dupraz, P., Cottet, S., Hamburger, F., Dolci, W., Felley-Bosco, E., and Thorens, B. (2000) J. Biol. Chem. 275, 37672--37678). To assess the role of the Janus kinase/signal transducer and activator of transcription (STAT) pathway in IFN-gamma intracellular signaling, we stably overexpressed SOCS-1 (suppressor of cytokine signaling-1) in the beta cell line. We demonstrated that SOCS-1 suppressed cytokine-induced STAT-1 phosphorylation and increased cellular accumulation. This was accompanied by a suppression of the effect of IFN-gamma on: (i) reduction in insulin promoter-luciferase reporter gene transcription, (ii) decrease in insulin mRNA and peptide content, and (iii) suppression of glucose-stimulated insulin secretion. Furthermore, SOCS-1 also suppressed the cellular effects that require the combined presence of IL-1 beta and IFN-gamma: induction of nitric oxide production and apoptosis. Together our data demonstrate that IFN-gamma is responsible for the cytokine-induced defect in insulin gene expression and secretion and that this effect can be completely blocked by constitutive inhibition of the Janus kinase/STAT pathway.

Impaired expression of the inducible cAMP early repressor accounts for sustained adipose CREB activity in obesity.

OBJECTIVEIncrease in adipose cAMP response binding protein (CREB) activity promotes adipocyte dysfunction and systemic insulin resistance in obese mice. This is achieved by increasing the expression of activating transcription factor 3 (ATF3). In this study we investigated whether impaired expression of the inducible cAMP early repressor (ICER), a transcriptional antagonist of CREB, is responsible for the increased CREB activity in adipocytes of obese mice and humans.RESEARCH DESIGN AND METHODSTotal RNA and nuclear proteins were prepared from visceral adipose tissue (VAT) of human nonobese or obese subjects, and white adipose tissue (WAT) of C57Bl6-Rj mice that were fed with normal or high-fat diet for 16 weeks. The expression of genes was monitored by real-time PCR, Western blotting, and electromobility shift assays. RNA interference was used to silence the expression of Icer.RESULTSThe expression of Icer/ICER was reduced in VAT and WAT of obese humans and mice, respectively. Diminution of Icer/ICER was restricted to adipocytes and was accompanied by a rise of Atf3/ATF3 and diminution of Adipoq/ADIPOQ and Glut4/GLUT4. Silencing the expression of Icer in 3T3-L1 adipocytes mimicked the results observed in human and mice cells and hampered glucose uptake, thus confirming the requirement of Icer for appropriate adipocyte function.CONCLUSIONSImpaired expression of ICER contributes to elevation in CREB target genes and, therefore, to the development of insulin resistance in obesity.

Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines.

Connexin36 (Cx36) is specifically expressed in neurons and in pancreatic beta-cells. Cx36 functions as a critical regulator of insulin secretion and content in beta-cells. In order to identify the molecular mechanisms that control the beta-cell expression of Cx36, we initiated the characterization of the human 5' regulatory region of the CX36 gene. A 2043-bp fragment of the human CX36 promoter was identified from a human BAC library and fused to a luciferase reporter gene. This promoter region was sufficient to confer specific expression to the reporter gene in insulin-secreting cell lines. Within this 5' regulatory region, a putative neuron-restrictive silencer element conserved between rodent and human species was recognized and binds the neuron-restrictive silencing factor (NRSF/REST). This factor is not expressed in insulin-secreting cells and neurons; it functions as a potent repressor through the recruitment of histone deacetylase to the promoter of neuronal genes. The NRSF-mediated repression of Cx36 in HeLa cells was abolished by trichostatin A, confirming the functional importance of histone deacetylase activity. Ectopic expression, by viral gene transfer, of NRSF/REST in different insulin-secreting beta-cell lines induced a marked reduction in Cx36 mRNA and protein content. Moreover, mutations in the Cx36 neuron-restrictive silencer element relieved the low transcriptional activity of the human CX36 promoter observed in HeLa cells and in INS-1 cells expressing NRSF/REST. The data showed that cx36 gene expression in insulin-producing beta-cell lines is strictly controlled by the transcriptional repressor NRSF/REST indicating that Cx36 participates to the neuronal phenotype of the pancreatic beta-cells.

CREB-2, a cellular CRE-dependent transcription repressor, functions in association with Tax as an activator of the human T-cell leukemia virus type 1 promoter.

The Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) has been implicated in human T-cell immortalization. The primary function of Tax is to transcriptionally activate the HTLV-1 promoter, but Tax is also known to stimulate expression of cellular genes. It has been reported to associate with several transcription factors, as well as proteins not involved in transcription. To better characterize potential cellular targets of Tax present in infected cells, a Saccharomyces cerevisiae two-hybrid screening was performed with a cDNA library constructed from the HTLV-1-infected MT2 cell line. From this study, we found 158 positive clones representing seven different cDNAs. We focused our attention on the cDNA encoding the transcription factor CREB-2. CREB-2 is an unconventional member of the ATF/CREB family in that it lacks a protein kinase A (PKA) phosphorylation site and has been reported to negatively regulate transcription from the cyclic AMP response element of the human enkephalin promoter. In this study, we demonstrate that CREB-2 cooperates with Tax to enhance viral transcription and that its basic-leucine zipper C-terminal domain is required for both in vitro and in vivo interactions with Tax. Our results confirm that the activation of the HTLV-1 promoter through Tax and factors of the ATF/CREB family is PKA independent.

The transcriptional repressor REST determines the cell-specific expression of the human MAPK8IP1 gene encoding IB1 (JIP-1).

Islet-brain 1 (IB1) is the human and rat homologue of JIP-1, a scaffold protein interacting with the c-Jun amino-terminal kinase (JNK). IB1 expression is mostly restricted to the endocrine pancreas and to the central nervous system. Herein, we explored the transcriptional mechanism responsible for this preferential islet and neuronal expression of IB1. A 731-bp fragment of the 5' regulatory region of the human MAPK8IP1 gene was isolated from a human BAC library and cloned upstream of a luciferase reporter gene. This construct drove high transcriptional activity in both insulin-secreting and neuron-like cells but not in unrelated cell lines. Sequence analysis of this promoter region revealed the presence of a neuron-restrictive silencer element (NRSE) known to bind repressor zinc finger protein REST. This factor is not expressed in insulin-secreting and neuron-like cells. By mobility shift assay, we confirmed that REST binds to the NRSE present in the IB1 promoter. Once transiently transfected in beta-cell lines, the expression vector encoding REST repressed IB1 transcriptional activity. The introduction of a mutated NRSE in the 5' regulating region of the IB1 gene abolished the repression activity driven by REST in insulin-secreting beta cells and relieved the low transcriptional activity of IB1 observed in unrelated cells. Moreover, transfection in non-beta and nonneuronal cell lines of an expression vector encoding REST lacking its transcriptional repression domain relieved IB1 promoter activity. Last, the REST-mediated repression of IB1 could be abolished by trichostatin A, indicating that deacetylase activity is required to allow REST repression. Taken together, these data establish a critical role for REST in the control of the tissue-specific expression of the human IB1 gene.

The TetR-type MfsR protein of the integrative and conjugative element (ICE) ICEclc controls both a putative efflux system and initiation of ICE transfer.

Integrative and conjugating elements (ICE) are self-transferable DNAs widely present in bacterial genomes, which often carry a variety of auxiliary genes of potential adaptive benefit. One of the model ICE is ICEclc, an element originally found in Pseudomonas knackmussii B13 and known for its propensity to provide its host with the capacity to metabolize chlorocatechols and 2-aminophenol. In this work, we studied the mechanism and target of regulation of MfsR, a TetR-type repressor previously found to exert global control on ICEclc horizontal transfer. By using a combination of ICEclc mutant and transcriptome analysis, gene reporter fusions, and DNA binding assays, we found that MfsR is a repressor of both its own expression and that of a gene cluster putatively coding for a major facilitator superfamily efflux system on ICEclc (named mfsABC). Phylogenetic analysis suggests that mfsR was originally located immediately adjacent to the efflux pump genes but became displaced from its original cis target DNA by a gene insertion. This resulted in divergence of the original bidirectional promoters into two separated individual regulatory units. Deletion of mfsABC did not result in a strong phenotype, and despite screening a large number of compounds and conditions, we were unable to define the precise current function or target of the putative efflux pump. Our data reconstruct how the separation of an ancestor mfsR-mfsABC system led to global control of ICEclc transfer by MfsR.

Posttranscriptional repression of GacS/GacA-controlled genes by the RNA-binding protein RsmE acting together with RsmA in the biocontrol strain Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium Pseudomonas fluorescens CHA0, the production of biocontrol factors (antifungal secondary metabolites and exoenzymes) is controlled at a posttranscriptional level by the GacS/GacA signal transduction pathway involving RNA-binding protein RsmA as a key regulatory element. This protein is assumed to bind to the ribosome-binding site of target mRNAs and to block their translation. RsmA-mediated repression is relieved at the end of exponential growth by two GacS/GacA-controlled regulatory RNAs RsmY and RsmZ, which bind and sequester the RsmA protein. A gene (rsmE) encoding a 64-amino-acid RsmA homolog was identified and characterized in strain CHA0. Overexpression of rsmE strongly reduced the expression of target genes (hcnA, for a hydrogen cyanide synthase subunit; aprA, for the main exoprotease; and phlA, for a component of 2,4-diacetylphloroglucinol biosynthesis). Single null mutations in either rsmA or rsmE resulted in a slight increase in the expression of hcnA, aprA, and phlA. By contrast, an rsmA rsmE double mutation led to strongly increased and advanced expression of these target genes and completely suppressed a gacS mutation. Both the RsmE and RsmA levels increased with increasing cell population densities in strain CHA0; however, the amount of RsmA showed less variability during growth. Expression of rsmE was controlled positively by GacA and negatively by RsmA and RsmE. Mobility shift assays demonstrated specific binding of RsmE to RsmY and RsmZ RNAs. The transcription and stability of both regulatory RNAs were strongly reduced in the rsmA rsmE double mutant. In conclusion, RsmA and RsmE together account for maximal repression in the GacS/GacA cascade of strain CHA0.

p21 as a transcriptional co-repressor of S-phase and mitotic control genes.

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes.

MAP/ERK kinase kinase 1 (MEKK1) mediates transcriptional repression by interacting with polycystic kidney disease-1 (PKD1) promoter-bound p53 tumor suppressor protein.

Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.

The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, when deprived of oxygen, generates ATP from arginine catabolism by enzymes of the arginine deiminase pathway, encoded by the arcDABC operon. Under conditions of low oxygen tension, the transcriptional activator ANR binds to a site centered 41.5 bp upstream of the arcD transcriptional start. ANR-mediated anaerobic induction was enhanced two- to threefold by extracellular arginine. This arginine effect depended, in trans, on the transcriptional regulator ArgR and, in cis, on an ArgR binding site centered at -73.5 bp in the arcD promoter. Binding of purified ArgR protein to this site was demonstrated by electrophoretic mobility shift assays and DNase I footprinting. This ArgR recognition site contained a sequence, 5'-TGACGC-3', which deviated in only 1 base from the common sequence motif 5'-TGTCGC-3' found in other ArgR binding sites of P. aeruginosa. Furthermore, an alignment of all known ArgR binding sites confirmed that they consist of two directly repeated half-sites. In the absence of ANR, arginine did not induce the arc operon, suggesting that ArgR alone does not activate the arcD promoter. According to a model proposed, ArgR makes physical contact with ANR and thereby facilitates initiation of arc transcription.