Induction of the acyl-coenzyme A synthetase gene by fibrates and fatty acids is mediated by a peroxisome proliferator response element in the C promoter.

The long-chain acyl-coenzyme A synthetase (ACS) gene gives rise to three transcripts containing different first exons preceded by specific regulatory regions A, B, and C. Exon-specific oligonucleotide hybridization indicated that only A-ACS mRNA is expressed in rat liver. Fibrate administration induced liver C-ACS strongly and A-ACS mRNA to a lesser extent. B-ACS mRNA remained undetectable. In primary rat hepatocytes and Fa-32 hepatoma cells C-ACS mRNA increased after treatment with fenofibric acid, alpha-bromopalmitate, tetradecylthioacetic acid, or alpha-linolenic acid. Nuclear run-on experiments indicated that fenofibric acid and alpha-bromopalmitate act at the transcriptional level. Transient transfections showed a 3.4-, 2.3-, and 2.2-fold induction of C-ACS promoter activity after fenofibric acid, alpha-bromopalmitate, and tetradecylthioacetic acid, respectively. Unilateral deletion and site-directed mutagenesis identified a peroxisome proliferator activator receptor (PPAR)-responsive element (PPRE) mediating the responsiveness to fibrates and fatty acids. This ACS PPRE contains three imperfect half sites spaced by 1 and 3 oligonucleotides and binds PPAR.retinoid X receptor heterodimers in gel retardation assays. In conclusion, the regulation of C-ACS mRNA expression by fibrates and fatty acids is mediated by PPAR.retinoid X receptor heterodimers interacting through a PPRE in the C-ACS promoters. PPAR therefore occupies a key position in the transcriptional control of a pivotal enzyme controlling the channeling of fatty acids into various metabolic pathways.

RNA-based gene duplication sheds new light on mammalian sex chromosome evolution

ABSTRACT : Gene duplication is a fundamental source of raw material for the origin of genetic novelty. It has been assumed for a long time that DNA-based gene duplication was the only source of new genes. Recently however, RNA-based gene duplication (retroposition) was shown in multiple organisms to contribute significantly to their genetic diversity. This mechanism produces intronless gene copies (retrocopies) that are inserted in random genomic position, independent of the position of the parental source genes. In human, mouse and fruit fly, it was demonstrated that the X-linked genes spawned an excess of functional retroposed gene copies (retrogenes). In human and mouse, the X chromosome also recruited an excess of retrogenes. Here we further characterized these interesting biases related to the X chromosome in mammals. Firstly, we have confirmed presence of the aforementioned biases in dog and opossum genome. Then based on the expression profile of retrogenes during various spermatogenetic stages, we have provided solid evidence that meiotic sex chromosome inactivation (MSCI) is responsible for an excess of retrogenes stemming from the X chromosome. Moreover, we showed that the X-linked genes started to export an excess of retrogenes just after the split of eutherian and marsupial mammalian lineages. This suggests that MSCI has originated around this time as well. More fundamentally, as MSCI reflects the spread of recombination barrier between the X and Y chromosomes during their evolution, our observation allowed us to re-estimate the age of mammalian sex chromosomes. Previous estimates suggested that they emerged in the common ancestor of all mammals (before the split of monotreme lineage); whereas, here we showed that they originated around the split of marsupial and eutherian lineages, after the divergence of monotremes. Thus, the therian (marsupial and eutherian) sex chromosomes are younger than previously thought. Thereafter, we have characterized the bias related to the recruitment of genes to the X chromosome. Sexually antagonistic forces are most likely driving this pattern. Using our limited retrogenes expression data, it is difficult to determine the exact nature of these forces but some conclusions have been made. Lastly, we looked at the history of this biased recruitment: it commenced around the split of marsupial and eutherian lineages (akin to the biased export of genes out of the X). In fact, the sexually antagonistic forces are predicted to appear just around that time as well. Thereby, the history of the recruitment of genes to the X, provides an indirect evidence that these forces are responsible for this bias.

Suppression of short interfering RNA-mediated gene silencing by the structural proteins of hepatitis C virus.

Viruses have evolved strategies to overcome the antiviral effects of the host at different levels. Besides specific defence mechanisms, the host responds to viral infection via the interferon pathway and also by RNA interference (RNAi). However, several viruses have been identified that suppress RNAi. We addressed the question of whether hepatitis C virus (HCV) suppresses RNAi, using cell lines constitutively expressing green fluorescent protein (GFP) and inducibly expressing HCV proteins. It was found that short interfering RNA-mediated GFP gene silencing was inhibited when the entire HCV polyprotein was expressed. Further studies showed that HCV structural proteins, and in particular envelope protein 2 (E2), were responsible for this inhibition. Co-precipitation assays demonstrated that E2 bound to Argonaute-2 (Ago-2), a member of the RNA-induced silencing complex, RISC. Thus, HCV E2 that interacts with Ago-2 is able to suppress RNAi.

RNA-based gene duplication: mechanistic and evolutionary insights.

Gene copies that stem from the mRNAs of parental source genes have long been viewed as evolutionary dead-ends with little biological relevance. Here we review a range of recent studies that have unveiled a significant number of functional retroposed gene copies in both mammalian and some non-mammalian genomes. These studies have not only revealed previously unknown mechanisms for the emergence of new genes and their functions but have also provided fascinating general insights into molecular and evolutionary processes that have shaped genomes. For example, analyses of chromosomal gene movement patterns via RNA-based gene duplication have shed fresh light on the evolutionary origin and biology of our sex chromosomes.

Regulation of c-fos expression by RNA polymerase elongation competence.

The molecular mechanisms underlying transcription elongation and their role in gene regulation are poorly characterized in eukaryotes. A number of genes, however, have been proposed to be regulated at the level of transcription elongation, including c-myc, c-fos and c-myb. Here, we analyze the control of transcription elongation at the mouse c-fos gene at the nucleotide level in intact cells. We find that RNA polymerases are engaged in the promoter-proximal part of the gene in the absence of gene activation signals and mRNA synthesis. Importantly, we determine that the engaged RNA polymerases originate from a continuous initiation of transcription which, in the absence of gene activation signals, terminate close to the promoter. We also observe that the c-fos gene presents an active chromatin conformation, with the promoter and upstream regulatory sequences constitutively occupied by proteins, accounting for the continuous initiation of RNA polymerase complexes. We propose that activation of c-fos gene expression results primarily from the assembly of elongation-competent RNA polymerases that can transcribe the complete gene. Our results suggest that the engaged RNA polymerases found downstream of a number of other eukaryotic promoters may be associated with transcription termination of elongation-incompetent polymerases in the absence of activating signals.

Characterisation of microbial communities colonising the hyphal surfaces of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are symbiotic soil fungi that are intimately associated with the roots of the majority of land plants. They colonise the interior of the roots and the hyphae extend into the soil. It is well known that bacterial colonisation of the rhizosphere can be crucial for many pathogenic as well as symbiotic plant-microbe interactions. However, although bacteria colonising the extraradical AMF hyphae (the hyphosphere) might be equally important for AMF symbiosis, little is known regarding which bacterial species would colonise AMF hyphae. In this study, we investigated which bacterial communities might be associated with AMF hyphae. As bacterial-hyphal attachment is extremely difficult to study in situ, we designed a system to grow AMF hyphae of Glomus intraradices and Glomus proliferum and studied which bacteria separated from an agricultural soil specifically attach to the hyphae. Characterisation of attached and non-attached bacterial communities was performed using terminal restriction fragment length polymorphism and clone library sequencing of 16S ribosomal RNA (rRNA) gene fragments. For all experiments, the composition of hyphal attached bacterial communities was different from the non-attached communities, and was also different from bacterial communities that had attached to glass wool (a non-living substratum). Analysis of amplified 16S rRNA genes indicated that in particular bacteria from the family of Oxalobacteraceae were highly abundant on AMF hyphae, suggesting that they may have developed specific interactions with the fungi.

Evolution of mRNA expression levels of autosomal and sex-linked genes in amniotes

In addition to differences in protein-coding gene sequences, changes in expression resulting from mutations in regulatory sequences have long been hypothesized to be responsible for phenotypic differences between species. However, unlike comparison of genome sequences, few studies, generally restricted to pairwise comparisons of closely related mammalian species, have assessed between-species differences at the transcriptome level. They reported that gene expression evolves at different rates in various organs and in a pattern that is overall consistent with neutral models of evolution. In the first part of my thesis, I investigated the evolution of gene expression in therian mammals (i.e.7 placental and marsupials), based on microarray data from human, mouse and the gray short-tailed opossum (Monodelphis domestica). In addition to autosomal genes, a special focus was given to the evolution of X-linked genes. The therian X chromosome was recently shown to be younger than previously thought and to harbor a specific gene content (e.g., genes involved in brain or reproductive functions) that is thought to have been shaped by specific sex-related evolutionary forces. Sex chromosomes derive from ordinary autosomes and their differentiation led to the degeneration of the Y chromosome (in mammals) or W chromosome (in birds). Consequently, X- or Z-linked genes differ in gene dose between males and females such that the heterogametic sex has half the X/Z gene dose compared to the ancestral state. To cope with this dosage imbalance, mammals have been reported to have evolved mechanisms of dosage compensation.¦In the first project, I could first show that transcriptomes evolve at different rates in different organs. Out of the five tissues I investigated, the testis is the most rapidly evolving organ at the gene expression level while the brain has the most conserved transcriptome. Second, my analyses revealed that mammalian gene expression evolution is compatible with a neutral model, where the rates of change in gene expression levels is linked to the efficiency of purifying selection in a given lineage, which, in turn, is determined by the long-term effective population size in that lineage. Thus, the rate of DNA sequence evolution, which could be expected to determine the rate of regulatory sequence change, does not seem to be a major determinant of the rate of gene expression evolution. Thus, most gene expression changes seem to be (slightly) deleterious. Finally, X-linked genes seem to have experienced elevated rates of gene expression change during the early stage of X evolution. To further investigate the evolution of mammalian gene expression, we generated an extensive RNA-Seq gene expression dataset for nine mammalian species and a bird. The analyses of this dataset confirmed the patterns previously observed with microarrays and helped to significantly deepen our view on gene expression evolution.¦In a specific project based on these data, I sought to assess in detail patterns of evolution of dosage compensation in amniotes. My analyses revealed the absence of male to female dosage compensation in monotremes and its presence in marsupials and, in addition, confirmed patterns previously described for placental mammals and birds. I then assessed the global level of expression of X/Z chromosomes and contrasted this with its ancestral gene expression levels estimated from orthologous autosomal genes in species with non-homologous sex chromosomes. This analysis revealed a lack of up-regulation for placental mammals, the level of expression of X-linked genes being proportional to gene dose. Interestingly, the ancestral gene expression level was at least partially restored in marsupials as well as in the heterogametic sex of monotremes and birds. Finally, I investigated alternative mechanisms of dosage compensation and found that gene duplication did not seem to be a widespread mechanism to restore the ancestral gene dose. However, I could show that placental mammals have preferentially down-regulated autosomal genes interacting with X-linked genes which underwent gene expression decrease, and thus identified a novel alternative mechanism of dosage compensation.

Questioning the utility of RNA-DNA chimera in gene repair.

In principle, we should be glad that Eric Kmiec and his colleagues published in Science's STKE (1) a detailed experimental protocol of their gene repair method (2, 3). However, a careful reading of their contribution raises more doubts about the method. The research published in Science five years ago by Kmiec and his colleagues was said to demonstrate that chimeric RNA-DNA oligonucleotides could correct the mutation responsible for sickle cell anemia with 50% efficiency (4). Such a remarkable result prompted many laboratories to attempt to replicate the research or utilize the method on their own systems. However, if the method worked at all, which it rarely did, the achieved efficiency was usually lower by several orders of magnitude. Now, in the Science's STKE protocol, we are given crucial information about the method and why it is so important to utilize these expensive chimeric RNA-DNA constructs. In the introduction we are told that the RNA-DNA duplex is more stable than a DNA-DNA duplex and so extends the half-life of the complexes formed between the targeted DNA and the chimeric RNA-DNA oligonucleotides. This logical explanation, however, conflicts with the statement in the section entitled "Transfection with Oligonucleotides and Plasmid DNA" that Kmiec and colleagues have recently demonstrated that classical single-stranded DNA oligonucleotides with a few protective phosphothioate linkages have a "gene repair conversion frequency rivaling that of the RNA/DNA chimera". Indeed, the research cited for that result actually states that single-stranded DNA oligonucleotides are in fact several-fold more efficient (3.7-fold) than the RNA-DNA chimeric constructs (5). If that is the case, it raises the question of why Kmiec and colleagues emphasize the importance of the RNA in their original chimeric constructs. Their own new results show that modified single-stranded DNA oligonucleotides are more effective than the expensive RNA-DNA hybrids. Moreover, the current efficiency of the gene repair by RNA-DNA hybrids, according to Kmiec and colleagues in their recent paper is only 4×10-4 even after several hours of pre-selection permitting multiplification of bacterial cells with the corrected plasmid (5). This efficiency is much lower than the 50% value reported five years ago, but is assuredly much closer to the reality.

Visualization of RNA polymerase II ternary transcription complexes formed in vitro on a Xenopus laevis vitellogenin gene.

Stable ternary transcription complexes assembled in vitro, using a HeLa whole-cell extract, have been isolated and visualized by electron microscopy. The formation of these stable complexes on the DNA fragment used as template, the 5' end region of the Xenopus laevis vitellogenin gene B2, depends on factors present in the whole-cell extract, RNA polymerase II and at least two nucleotides. Interestingly, bending in the DNA fragment was frequently observed at the binding site of RNA polymerase II. Dinucleotides that can prime initiation within a short sequence of approximately 10 contiguous nucleotides centered around the initiation site used in vivo, also favour the formation of stable complexes. In addition, pre-initiation complexes were isolated and it was shown that factors in the extract involved in their formation are more abundant than the RNA polymerase II molecules available for binding. The possible implication of this observation relative to the in vivo situation is discussed.

Genomic Study of RNA Polymerase II and III SNAP(c)-Bound Promoters Reveals a Gene Transcribed by Both Enzymes and a Broad Use of Common Activators.

SNAP(c) is one of a few basal transcription factors used by both RNA polymerase (pol) II and pol III. To define the set of active SNAP(c)-dependent promoters in human cells, we have localized genome-wide four SNAP(c) subunits, GTF2B (TFIIB), BRF2, pol II, and pol III. Among some seventy loci occupied by SNAP(c) and other factors, including pol II snRNA genes, pol III genes with type 3 promoters, and a few un-annotated loci, most are primarily occupied by either pol II and GTF2B, or pol III and BRF2. A notable exception is the RPPH1 gene, which is occupied by significant amounts of both polymerases. We show that the large majority of SNAP(c)-dependent promoters recruit POU2F1 and/or ZNF143 on their enhancer region, and a subset also recruits GABP, a factor newly implicated in SNAP(c)-dependent transcription. These activators associate with pol II and III promoters in G1 slightly before the polymerase, and ZNF143 is required for efficient transcription initiation complex assembly. The results characterize a set of genes with unique properties and establish that polymerase specificity is not absolute in vivo.

Feasibility of RNA studies on illegitimate transcription for molecular characterization of splicing mutations in the ATP7B gene: a case report.

Approximately 520 Wilson disease-causing mutations in the ATP7B gene have been described to date. In this study we report DNA and RNA analyses carried out for molecular characterization of a consensus sequence splicing mutation found in homozygosity in a Swiss Wilson disease patient. RNA analysis of 1946 +6 T→C in both the peripheral lymphoblasts and liver resulted in the production in the propositus of only an alternative transcript lacking exons 6, 7, and 8 resulting most likely in alterations of cell biochemistry and disease. The patient presents an early form of severe hepatic disease characterized by hepatosplenomegaly, reduced hepatic function, anemia and thrombocytopenia indicating that 1946 +6 T→C is a severe mutation. Since identical results were obtained from both peripheral lymphoblasts and liver they also suggest that RNA studies of illegitimate transcripts can be safely used for molecular characterization of ATP7B splicing mutations, thus improving genetic counseling and diagnosis of Wilson disease. Moreover these studies, contribute to reveal the exact molecular mechanisms producing Wilson disease.

Interleukin-1 receptor antagonist gene polymorphism and circulating levels of human immunodeficiency virus type 1 RNA in Brazilian women.

Interleukin-1 receptor antagonist (IL-1ra) gene polymorphisms in 83 human immunodeficiency virus (HIV)-seropositive women were evaluated. Fourteen of the subjects (16.9%) were homozygous for IL-1ra allele 2 (IL-1RN*2). These women had a lower median level of HIV RNA than did women homozygous for allele 1 (IL-1RN*1) (P = 0.01) or heterozygous for both alleles (P = 0.04). Among 46 subjects not receiving antiretroviral treatment, HIV levels were also reduced in IL-1RN*2 homozygous individuals (P < 0.05). There was no relation between IL-1ra alleles and CD4 levels.

Phosphorylation by casein kinase 2 facilitates rRNA gene transcription by promoting dissociation of TIF-IA from elongating RNA polymerase I.

The protein kinase casein kinase 2 (CK2) phosphorylates different components of the RNA polymerase I (Pol I) transcription machinery and exerts a positive effect on rRNA gene (rDNA) transcription. Here we show that CK2 phosphorylates the transcription initiation factor TIF-IA at serines 170 and 172 (Ser170/172), and this phosphorylation triggers the release of TIF-IA from Pol I after transcription initiation. Inhibition of Ser170/172 phosphorylation or covalent tethering of TIF-IA to the RPA43 subunit of Pol I inhibits rDNA transcription, leading to perturbation of nucleolar structure and cell cycle arrest. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrate that dissociation of TIF-IA from Pol I is a prerequisite for proper transcription elongation. In support of phosphorylation of TIF-IA switching from the initiation into the elongation phase, dephosphorylation of Ser170/172 by FCP1 facilitates the reassociation of TIF-IA with Pol I, allowing a new round of rDNA transcription. The results reveal a mechanism by which the functional interplay between CK2 and FCP1 sustains multiple rounds of Pol I transcription.

Gene transfer engineering for astrocyte-specific silencing in the CNS.

Cell-type-specific gene silencing is critical to understand cell functions in normal and pathological conditions, in particular in the brain where strong cellular heterogeneity exists. Molecular engineering of lentiviral vectors has been widely used to express genes of interest specifically in neurons or astrocytes. However, we show that these strategies are not suitable for astrocyte-specific gene silencing due to the processing of small hairpin RNA (shRNA) in a cell. Here we develop an indirect method based on a tetracycline-regulated system to fully restrict shRNA expression to astrocytes. The combination of Mokola-G envelope pseudotyping, glutamine synthetase promoter and two distinct microRNA target sequences provides a powerful tool for efficient and cell-type-specific gene silencing in the central nervous system. We anticipate our vector will be a potent and versatile system to improve the targeting of cell populations for fundamental as well as therapeutic applications.

Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.