LEDGF/p75 TATA-less promoter is driven by the transcription factor Sp1.

PSIP1 (PC4 and SFRS1 interacting protein 1) encodes two splice variants: lens epithelium-derived growth factor or p75 (LEDGF/p75) and p52. PSIP1 gene products were shown to be involved in transcriptional regulation, affecting a plethora of cellular processes, including cell proliferation, cell survival, and stress response. Furthermore, LEDGF/p75 has implications for various diseases and infections, including autoimmunity, leukemia, embryo development, psoriasis, and human immunodeficiency virus integration. Here, we reported the first characterization of the PSIP1 promoter. Using 5' RNA ligase-mediated rapid amplification of cDNA ends, we identified novel transcription start sites in different cell types. Using a luciferase reporter system, we identified regulatory elements controlling the expression of LEDGF/p75 and p52. These include (i) minimal promoters (-112/+59 and +609/+781) that drive the basal expression of LEDGF/p75 and of the shorter splice variant p52, respectively; (ii) a sequence (+319/+397) that may control the ratio of LEDGF/p75 expression to p52 expression; and (iii) a strong enhancer (-320/-207) implicated in the modulation of LEDGF/p75 transcriptional activity. Computational, biochemical, and genetic approaches enabled us to identify the transcription factor Sp1 as a key modulator of the PSIP1 promoter, controlling LEDGF/p75 transcription through two binding sites at -72/-64 and -46/-36. Overall, our results provide initial data concerning LEDGF/p75 promoter regulation, giving new insights to further understand its biological function and opening the door for new therapeutic strategies in which LEDGF/p75 is involved.

The jasmonate pathway.

Plants are faced with many of the same problems as animals-a need for regulation of metabolic processes and reproduction and for defense against enemies. Jasmonates in plants serve key roles in gene and metabolic regulation, defense, responses to trauma, reproduction, and possibly communication. Some remarkable features of plant responses, such as production of repellent volatiles as a defense against herbivorous insects, or the massive transcriptional reprogramming that occurs in response to wounding, are under the control of the jasmonate pathway. Details of the jasmonate signaling pathway are currently at the center of active research that is generating exciting results. The Jasmonate Biochemical Pathway at the STKE Connections Maps is designed to present and keep pace with these developments.

Mouse interleukin-2 receptor alpha gene expression. Delimitation of cis-acting regulatory elements in transgenic mice and by mapping of DNase-I hypersensitive sites.

The alpha chain of the interleukin-2 receptor (IL-2R alpha) is a key regulator of lymphocyte proliferation. To analyze the mechanisms controlling its expression in normal cells, we used the 5'-flanking region (base pairs -2539/+93) of the mouse gene to drive chloramphenicol acetyltransferase expression in four transgenic mouse lines. Constitutive transgene activity was restricted to lymphoid organs. In mature T lymphocytes, transgene and endogenous IL-2R alpha gene expression was stimulated by concanavalin A and up-regulated by IL-2 with very similar kinetics. In thymic T cell precursors, IL-1 and IL-2 cooperatively induced transgene and IL-2R alpha gene expression. These results show that regulation of the endogenous IL-2R alpha gene occurs mainly at the transcriptional level. They demonstrate that cis-acting elements in the 5'-flanking region present in the transgene confer correct tissue specificity and inducible expression in mature T cells and their precursors in response to antigen, IL-1, and IL-2. In a complementary approach, we screened the 5' end of the endogenous IL-2R alpha gene for DNase-I hypersensitive sites. We found three lymphocyte specific DNase-I hypersensitive sites. Two, at -0.05 and -5.3 kilobase pairs, are present in resting T cells. A third site appears at -1.35 kilobase pairs in activated T cells. It co-localizes with IL-2-responsive elements identified by transient transfection experiments.

Regulation of horizontal gene transfer of the catabolic island ICEclc

ICEclc is a mobile genetic element found in two copies on the chromosome of the bacterium Pseudomonas knackmussii B13. ICEclc harbors genes encoding metabolic pathways for the degradation of chlorocatechols (CLC) and 2-aminophenol (2AP). At low frequencies, ICEclc excises from the chromosome, closes into a circular DNA molecule which can transfer to another bacterium via conjugation. Once in the recipient cell, ICEclc can reintegrate into the chromosome by site-specific recombination. This thesis aimed at identifying the regulatory network underlying the decisions for ICEclc horizontal transfer (HGT). The first chapter is an introduction on integrative and conjugative elements (ICEs) more in general, of which ICEclc is one example. In particular I emphasized the current knowledge of regulation and conjugation machineries of the different classes of ICE. In the second chapter, I describe a transcriptional analysis using microarrays and other experiments to understand expression of ICEclc in exponential and stationary phase. By overlaying transcriptomic profiles with Northern hybridizations and RT- PCR data, we established a transcription map for the entire core region of ICEclc, a region assumed to encode the ICE conjugation process. We also demonstrated how transcription of the ICEclc core is maximal in stationary phase, which correlates to expression of reporter genes fused to key ICEclc promoters. In the third chapter, I present a transcriptome analysis of ICEclc in a variety of different host species, in order to explore whether there are species-specific differences. In the fourth chapter, I focus on the role of a curious ICEclc-encoded TetR-type transcriptional repressor. We find that this gene, which we name mfsR, not only controls its own expression but that of a set of genes for a putative multi-drug efflux pump (mfsABC) as well. By using a combination of biochemical and molecular biology techniques, I could show that MfsR specifically binds to operator boxes in two ICEclc promoters (PmfsR and PmfsA), inhibiting the transcription of both the mfsR and mfsABC-orf38184 operons. Although we could not detect a clear phenotype of an mfsABC deletion, we discuss the implications of pump gene reorganizations in ICEclc and close relatives. In the fifth chapter, we find that mfsR not only controls its own expression and that of the mfsABC operon, but is also indirectly controlling ICEclc transfer. Using gene deletions, microarrays, transfer assays and microscopy-based reporter fusions, we demonstrate that mfsR actually controls a small operon of three regulatory genes. The last gene of this mfsR operon, orf17162, encodes a LysR-type activator that when deleted strongly impairs ICEclc transfer. Interestingly, deletion of mfsR leads to transfer competence in almost all cells, thereby overruling the bistability process in the wild-type. In the final sixth chapter, I discuss the relevance of the present thesis and the resulting perspectives for future studies.

Circadian regulation of renal function.

Urinary excretion of water and all major electrolytes exhibit robust circadian oscillations. The 24-h periodicity has been well documented for several important determinants of urine formation, including renal blood flow, glomerular filtration, tubular reabsorption, and tubular secretion. Disturbance of the renal circadian rhythms is increasingly recognized as a risk factor for hypertension, polyuria, and other diseases and may contribute to renal fibrosis. The origin of these rhythms has been attributed to the reactive response of the kidney to circadian changes in volume and/or in the composition of extracellular fluids that are entrained by rest/activity and feeding/fasting cycles. However, numerous studies have shown that most of the renal excretory rhythms persist for long periods of time, even in the absence of periodic environmental cues. These observations led to the hypothesis of the existence of a self-sustained mechanism, enabling the kidney to anticipate various predictable circadian challenges to homeostasis. The molecular basis of this mechanism remained unknown until the recent discovery of the mammalian circadian clock made of a system of autoregulatory transcriptional/translational feedback loops, which have been found in all tissues studied, including the kidney. Here, we present a review of the growing evidence showing the involvement of the molecular clock in the generation of renal excretory rhythms.

Antifungal resistance and new strategies to control fungal infections.

Despite improvement of antifungal therapies over the last 30 years, the phenomenon of antifungal resistance is still of major concern in clinical practice. In the last 10 years the molecular mechanisms underlying this phenomenon were extensively unraveled. In this paper, after a brief overview of currently available antifungals, molecular mechanisms of antifungal resistance will be detailed. It appears that major mechanisms of resistance are essential due to the deregulation of antifungal resistance effector genes. This deregulation is a consequence of point mutations occurring in transcriptional regulators of these effector genes. Resistance can also follow the emergence of point mutations directly in the genes coding antifungal targets. In addition we further describe new strategies currently undertaken to discover alternative therapy targets and antifungals. Identification of new antifungals is essentially achieved by the screening of natural or synthetic chemical compound collections. Discovery of new putative antifungal targets is performed through genome-wide approaches for a better understanding of the human pathogenic fungi biology.

Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil.

The root-colonizing bacterium Pseudomonas fluorescens CHA0 was used to construct an oxygen-responsive biosensor. An anaerobically inducible promoter of Pseudomonas aeruginosa, which depends on the FNR (fumarate and nitrate reductase regulation)-like transcriptional regulator ANR (anaerobic regulation of arginine deiminase and nitrate reductase pathways), was fused to the structural lacZ gene of Escherichia coli. By inserting the reporter fusion into the chromosomal attTn7 site of P. fluorescens CHA0 by using a mini-Tn7 transposon, the reporter strain, CHA900, was obtained. Grown in glutamate-yeast extract medium in an oxystat at defined oxygen levels, the biosensor CHA900 responded to a decrease in oxygen concentration from 210 x 10(2) Pa to 2 x 10(2) Pa of O(2) by a nearly 100-fold increase in beta-galactosidase activity. Half-maximal induction of the reporter occurred at about 5 x 10(2) Pa. This dose response closely resembles that found for E. coli promoters which are activated by the FNR protein. In a carbon-free buffer or in bulk soil, the biosensor CHA900 still responded to a decrease in oxygen concentration, although here induction was about 10 times lower and the low oxygen response was gradually lost within 3 days. Introduced into a barley-soil microcosm, the biosensor could report decreasing oxygen concentrations in the rhizosphere for a 6-day period. When the water content in the microcosm was raised from 60% to 85% of field capacity, expression of the reporter gene was elevated about twofold above a basal level after 2 days of incubation, suggesting that a water content of 85% caused mild anoxia. Increased compaction of the soil was shown to have a faster and more dramatic effect on the expression of the oxygen reporter than soil water content alone, indicating that factors other than the water-filled pore space influenced the oxygen status of the soil. These experiments illustrate the utility of the biosensor for detecting low oxygen concentrations in the rhizosphere and other soil habitats.

Histone deacetylase inhibitors impair innate immune responses

SUMMARYThe innate immune system plays a central role in host defenses against invading pathogens. Innate immune cells sense the presence of pathogens through pattern recognition receptors that trigger intracellular signaling, leading to the production of pro-inflammatory mediators like cytokines, which shape innate and adaptive immune responses. Both by excess and by default inflammation may be detrimental to the host. Indeed, severe sepsis and septic shock are lethal complications of infections characterized by a dysregulated inflammatory response.In recent years, members of the superfamily of histone deacetylases have been the focus of great interest. In mammals, histone deacetylases are broadly classified into two main subfamilies comprising histone deacetylases 1-11 (HDAC1-11) and sirtuins 1-7 (SIRT1-7). These enzymes influence gene expression by deacetylating histones and numerous non-histone proteins. Histone deacetylases have been involved in the development of oncologic, metabolic, cardiovascular, neurodegenerative and autoimmune diseases. Pharmacological modulators of histone deacetylase activity, principally inhibitors, have been developed for the treatment of cancer and metabolic diseases. When we initiated this project, several studies suggested that inhibitors of HDAC 1-11 have anti-inflammatory activity. Yet, their influence on innate immune responses was largely uncharacterized. The present study was initiated to fill in this gap.In the first part of this work, we report the first comprehensive study of the effects of HDAC 1- 11 inhibitors on innate immune responses in vitro and in vivo. Strikingly, expression studies revealed that HDAC1-11 inhibitors act essentially as negative regulators of basal and microbial product- induced expression of critical immune receptors and antimicrobial products by mouse and human innate immune cells like macrophages and dendritic cells. Furthermore, we describe a new molecular mechanism whereby HDAC1-11 inhibitors repress pro-inflammatory cytokine expression through the induction of the expression and the activity of the transcriptional repressor Μί-2β. HDAC1-11 inhibitors also impair the potential of macrophages to engulf and kill bacteria. Finally, mice treated with an HDAC inhibitor are more susceptible to non-severe bacterial and fungal infection, but are protected against toxic and septic shock. Altogether these data support the concept that HDAC 1-11 inhibitors have potent anti-inflammatory and immunomodulatory activities in vitro and in vivo.Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays a central role in innate immune responses, cell proliferation and oncogenesis. In the second part of this manuscript, we demonstrate that HDAC1-11 inhibitors inhibit MIF expression in vitro and in vivo and describe a novel molecular mechanism accounting for these effects. We propose that inhibition of MIF expression by HDAC 1-11 inhibitors may contribute to the antitumorigenic and anti-inflammatory effects of these drugs.NAD+ is an essential cofactor of sirtuins activity and one of the major sources of energy within the cells. Therefore, sirtuins link deacetylation to NAD+ metabolism and energy status. In the last part of this thesis, we report preliminary results indicating that a pharmacological inhibitor of SIRT1-2 drastically decreases pro-inflammatory cytokine production (RNA and protein) and interferes with MAP kinase intracellular signal transduction pathway in macrophages. Moreover, administration of the SIRT1-2 inhibitor protects mice from lethal endotoxic shock and septic shock.Overall, our studies demonstrate that inhibitors of HDAC1-11 and sirtuins are powerful anti-inflammatory molecules. Given their profound negative impact on the host antimicrobial defence response, these inhibitors might increase the susceptibility to opportunistic infections, especially in immunocompromised cancer patients. Yet, these inhibitors might be useful to control the inflammatory response in severely ill septic patients or in patients suffering from chronic inflammatory diseases.

Promoter architecture of mouse olfactory receptor genes.

Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.

Isochorismate synthase (PchA), the first and rate-limiting enzyme in salicylate biosynthesis of Pseudomonas aeruginosa.

In Pseudomonas aeruginosa the extracellular metabolite and siderophore pyochelin is synthesized from two major precursors, chorismate and l-cysteine via salicylate as an intermediate. The regulatory role of isochorismate synthase, the first enzyme in the pyochelin biosynthetic pathway, was studied. This enzyme is encoded by pchA, the last gene in the pchDCBA operon. The PchA protein was purified to apparent electrophoretic homogeneity from a PchA-overexpressing P. aeruginosa strain. The native enzyme was a 52-kDa monomer in solution, and its activity strictly depended on Mg(2+). At pH 7.0, the optimum, a K(m) = 4.5 microm and a k(cat) = 43.1 min(-1) were determined for chorismate. No feedback inhibitors or other allosteric effectors were found. The intracellular PchA concentration critically determined the rate of salicylate formation both in vitro and in vivo. In cultures grown in iron-limiting media to high cell densities, overexpression of the pchA gene resulted in overproduction of salicylate as well as in enhanced pyochelin formation. From this work and earlier studies, it is proposed that one important factor influencing the flux through the pyochelin biosynthetic pathway is the PchA concentration, which is determined at a transcriptional level, with pyochelin acting as a positive signal and iron as a negative signal.

PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans.

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was approximately 35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-alpha, were recovered from AS (MTL-a/MTL-alpha), while a single type of PAP1 allele (PAP1-alpha) was recovered from AR isolates (MTL-alpha/MTL-alpha). Among the heterozygous deletions of PAP1-a (Deltapap1-a/PAP1-alpha) and PAP1-alpha (PAP1-a/Deltapap1-alpha), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

Peroxisome proliferator-activated receptors beta/delta: emerging roles for a previously neglected third family member.

PURPOSE OF REVIEW: Peroxisome proliferator-activated receptors alpha, beta/delta and gamma are members of the nuclear receptor superfamily. They mediate the effects of fatty acids and their derivatives at the transcriptional level, and are considered to be lipid sensors that participate in the regulation of energy homeostasis. Compared with the alpha and gamma peroxisome proliferator-activated receptor isotypes, peroxisome proliferator-activated receptor beta functions have long remained an enigma. In this review, we focus on emerging knowledge about peroxisome proliferator-activated receptor beta activation and roles. RECENT FINDINGS: We review recent data that suggest key roles in basic cell functions, such as proliferation, differentiation and survival, and in embryonic development and lipid metabolism in peripheral tissues. SUMMARY: The newly unveiled roles of peroxisome proliferator-activated receptor beta in important basic cell functions certainly justify a further exploration of its potential as a therapeutic target in pathologies such as metabolic syndrome X or skin diseases.

Neuronal traits are required for glucose-induced insulin secretion.

The transcriptional repressor RE1 silencer transcription factor (REST) is an important factor that restricts some neuronal traits to neurons. Since these traits are also present in pancreatic beta-cells, we evaluated their role by generating a model of insulin-secreting cells that express REST. The presence of REST led to a decrease in expression of its known target genes, whereas insulin expression and its cellular content were conserved. As a consequence of REST expression, the capacity to secrete insulin in response to mitochondrial fuels, a particularity of mature beta-cells, was impaired. These data provide evidence that REST target genes are required for an appropriate glucose-induced insulin secretion.

Deficient T cell fate specification in mice with an induced inactivation of Notch1.

Notch proteins are cell surface receptors that mediate developmental cell specification events. To explore the function of murine Notch1, an essential portion of the gene was flanked with loxP sites and inactivation induced via interferon-regulated Cre recombinase. Mice with a neonatally induced loss of Notch1 function were transiently growth retarded and had a severe deficiency in thymocyte development. Competitive repopulation of lethally irradiated wild-type hosts with wild-type- and Notch1-deficient bone marrow revealed a cell autonomous blockage in T cell development at an early stage, before expression of T cell lineage markers. Notch1-deficient bone marrow did, however, contribute normally to all other hematopoietic lineages. These findings suggest that Notch1 plays an obligatory and selective role in T cell lineage induction.

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.