Gènes, environnement et neurodéveloppement: te cas de la schizophrénie [Schizophrenia: genes, environment and neurodevelopment].

Psychoses are complex diseases resulting from the interaction between genetic vulnerability factors and various environmental risk factors during the brain development and leading to the emergence of the clinical phenotype at the end of adolescence. Among the mechanisms involved, a redox imbalance plays an important role, inducing oxidative stress damaging to developing neurons. As a consequence, the excitatory/inhibitory balance in cortex and the pathways connecting brain areas are both impaired. Childhood and adolescence appear as critical periods of vulnerability for deleterious environmental insults. Antioxidants, applied during the environmental impacts, should allow preventing these impairments as well as their clinical consequences.

Integration of genome-wide association studies with biological knowledge identifies six novel genes related to kidney function.

In conducting genome-wide association studies (GWAS), analytical approaches leveraging biological information may further understanding of the pathophysiology of clinical traits. To discover novel associations with estimated glomerular filtration rate (eGFR), a measure of kidney function, we developed a strategy for integrating prior biological knowledge into the existing GWAS data for eGFR from the CKDGen Consortium. Our strategy focuses on single nucleotide polymorphism (SNPs) in genes that are connected by functional evidence, determined by literature mining and gene ontology (GO) hierarchies, to genes near previously validated eGFR associations. It then requires association thresholds consistent with multiple testing, and finally evaluates novel candidates by independent replication. Among the samples of European ancestry, we identified a genome-wide significant SNP in FBXL20 (P = 5.6 × 10(-9)) in meta-analysis of all available data, and additional SNPs at the INHBC, LRP2, PLEKHA1, SLC3A2 and SLC7A6 genes meeting multiple-testing corrected significance for replication and overall P-values of 4.5 × 10(-4)-2.2 × 10(-7). Neither the novel PLEKHA1 nor FBXL20 associations, both further supported by association with eGFR among African Americans and with transcript abundance, would have been implicated by eGFR candidate gene approaches. LRP2, encoding the megalin receptor, was identified through connection with the previously known eGFR gene DAB2 and extends understanding of the megalin system in kidney function. These findings highlight integration of existing genome-wide association data with independent biological knowledge to uncover novel candidate eGFR associations, including candidates lacking known connections to kidney-specific pathways. The strategy may also be applicable to other clinical phenotypes, although more testing will be needed to assess its potential for discovery in general.

HLA-C cell surface expression and control of HIV/AIDS correlate with a variant upstream of HLA-C.

A variant 35 kb upstream of the HLA-C gene (-35C/T) was previously shown to associate with HLA-C mRNA expression level and steady-state plasma HIV RNA levels. We genotyped this variant in 1,698 patients of European ancestry with HIV. Individuals with known seroconversion dates were used for disease progression analysis and those with longitudinal viral load data were used for viral load analysis. We further tested cell surface expression of HLA-C in normal donors using an HLA-C-specific antibody. We show that the -35C allele is a proxy for high HLA-C cell surface expression, and that individuals with high-expressing HLA-C alleles progress more slowly to AIDS and control viremia significantly better than individuals with low HLA-C expressing alleles. These data strongly implicate high HLA-C expression levels in more effective control of HIV-1, potentially through better antigen presentation to cytotoxic T lymphocytes or recognition and killing of infected cells by natural killer cells.

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.

The final N-terminal trimming of a subaminoterminal proline-containing HLA class I-restricted antigenic peptide in the cytosol is mediated by two peptidases.

The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities.

Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.

According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions.

High-level molecular diversity of copper-zinc superoxide dismutase genes among and within species of arbuscular Mycorrhizal fungi.

In the ecologically important arbuscular mycorrhizal fungi (AMF), Sod1 encodes a functional polypeptide that confers increased tolerance to oxidative stress and that is upregulated inside the roots during early steps of the symbiosis with host plants. It is still unclear whether its expression is directed at scavenging reactive oxygen species (ROS) produced by the host, if it plays a role in the fungus-host dialogue, or if it is a consequence of oxidative stress from the surrounding environment. All these possibilities are equally likely, and molecular variation at the Sod1 locus can possibly have adaptive implications for one or all of the three mentioned functions. In this paper, we analyzed the diversity of the Sod1 gene in six AMF species, as well as 14 Glomus intraradices isolates from a single natural population. By sequencing this locus, we identified a large amount of nucleotide and amino acid molecular diversity both among AMF species and individuals, suggesting a rapid divergence of its codons. The Sod1 gene was monomorphic within each isolate we analyzed, and quantitative PCR strongly suggest this locus is present as a single copy in G. intraradices. Maximum-likelihood analyses performed using a variety of models for codon evolution indicated that a number of amino acid sites most likely evolved under the regime of positive selection among AMF species. In addition, we found that some isolates of G. intraradices from a natural population harbor very divergent orthologous Sod1 sequences, and our analysis suggested that diversifying selection, rather than recombination, was responsible for the persistence of this molecular diversity within the AMF population.

Specific Silencing of the REST Target Genes in Insulin-Secreting Cells Uncovers Their Participation in Beta Cell Survival.

The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival.

Ex vivo analysis of human antigen-specific CD8+ T-cell responses: quality assessment of fluorescent HLA-A2 multimer and interferon-gamma ELISPOT assays for patient immune monitoring.

The authors developed a standardized approach for immune monitoring of antigen-specific CD8+ T cells within peripheral blood lymphocytes (PBLs) that combines direct ex vivo analysis of Melan-A/MART-1 and influenza-specific CD8+ T cells with HLA-A2/peptide multimers and interferon-gamma ELISPOT assays. Here the authors assessed the quality of results obtained with 180 PBLs from healthy donors and melanoma patients. Reproducibility of the multimer assay was good (average of 15% variation). In the absence of in vivo antigen-specific T-cell responses, physiologic fluctuations of multimer-positive T cells was low, with variation coefficients of 20% for Melan-A and 28% for influenza-specific T cells. In contrast, patients with vaccination-induced T-cell responses had significantly increased T-cell frequencies clearly exceeding physiologic fluctuations. Comparable results were obtained with ELISPOT assays. In conclusion, this approach is well suited to assess T-cell responses as biologic endpoints in clinical vaccine studies.

Native homing endonucleases can target conserved genes in humans and in animal models.

In recent years, both homing endonucleases (HEases) and zinc-finger nucleases (ZFNs) have been engineered and selected for the targeting of desired human loci for gene therapy. However, enzyme engineering is lengthy and expensive and the off-target effect of the manufactured endonucleases is difficult to predict. Moreover, enzymes selected to cleave a human DNA locus may not cleave the homologous locus in the genome of animal models because of sequence divergence, thus hampering attempts to assess the in vivo efficacy and safety of any engineered enzyme prior to its application in human trials. Here, we show that naturally occurring HEases can be found, that cleave desirable human targets. Some of these enzymes are also shown to cleave the homologous sequence in the genome of animal models. In addition, the distribution of off-target effects may be more predictable for native HEases. Based on our experimental observations, we present the HomeBase algorithm, database and web server that allow a high-throughput computational search and assignment of HEases for the targeting of specific loci in the human and other genomes. We validate experimentally the predicted target specificity of candidate fungal, bacterial and archaeal HEases using cell free, yeast and archaeal assays.

Genome-wide association analysis of blood-pressure traits in African-ancestry individuals reveals common associated genes in African and non-African populations.

High blood pressure (BP) is more prevalent and contributes to more severe manifestations of cardiovascular disease (CVD) in African Americans than in any other United States ethnic group. Several small African-ancestry (AA) BP genome-wide association studies (GWASs) have been published, but their findings have failed to replicate to date. We report on a large AA BP GWAS meta-analysis that includes 29,378 individuals from 19 discovery cohorts and subsequent replication in additional samples of AA (n = 10,386), European ancestry (EA) (n = 69,395), and East Asian ancestry (n = 19,601). Five loci (EVX1-HOXA, ULK4, RSPO3, PLEKHG1, and SOX6) reached genome-wide significance (p < 1.0 × 10(-8)) for either systolic or diastolic BP in a transethnic meta-analysis after correction for multiple testing. Three of these BP loci (EVX1-HOXA, RSPO3, and PLEKHG1) lack previous associations with BP. We also identified one independent signal in a known BP locus (SOX6) and provide evidence for fine mapping in four additional validated BP loci. We also demonstrate that validated EA BP GWAS loci, considered jointly, show significant effects in AA samples. Consequently, these findings suggest that BP loci might have universal effects across studied populations, demonstrating that multiethnic samples are an essential component in identifying, fine mapping, and understanding their trait variability.

Meta-analysis of estrogen response in MCF-7 distinguishes early target genes involved in signaling and cell proliferation from later target genes involved in cell cycle and DNA repair.

ABSTRACT: BACKGROUND: Many studies have been published outlining the global effects of 17 beta-estradiol (E2) on gene expression in human epithelial breast cancer derived MCF-7 cells. These studies show large variation in results, reporting between ~100 and ~1500 genes regulated by E2, with poor overlap. RESULTS: We performed a meta-analysis of these expression studies, using the Rank product method to obtain a more accurate and stable list of the differentially expressed genes, and of pathways regulated by E2. We analyzed 9 time-series data sets, concentrating on response at 3-4 hrs (early) and at 24 hrs (late). We found >1000 statistically significant probe sets after correction for multiple testing at 3-4 hrs, and >2000 significant probe sets at 24 hrs. Differentially expressed genes were examined by pathway analysis. This revealed 15 early response pathways, mostly related to cell signaling and proliferation, and 20 late response pathways, mostly related to breast cancer, cell division, DNA repair and recombination. CONCLUSIONS: Our results show that meta-analysis identified more differentially expressed genes than the individual studies, and that these genes act together in networks. These results provide new insight into E2 regulated mechanisms, especially in the context of breast cancer.

Genes encoding tumor necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes.

Progressive destruction of the insulin-producing beta cells in nonobese diabetic mice is observed after infiltration of the pancreas with lymphocytes [Makino, S., Kunimoto, K., Muraoka, Y., Mizushima, Y., Katagiri, K. &amp; Tochino, Y. (1980) Exp. Anim. (Tokyo) 29, 1-13]. We show that the genes for tumor necrosis factor alpha and granzyme A, a serine protease associated with cytoplasmic granules of cytotoxic cells, are expressed during the development of spontaneous diabetes mellitus in the nonobese diabetic mouse. Granzyme A-positive cells are found both in and surrounding the islets, implying induction prior to islet infiltration. Tumor necrosis factor alpha expression is exclusively observed in the intra-islet infiltrate, predominantly in lymphocytes adjacent to insulin-producing beta cells, the targets of the autoimmune destruction, implying that tumor necrosis factor alpha expression is induced locally--i.e., in the islet. A considerable portion of cells expressing tumor necrosis factor alpha appear to be CD4+ T cells. This T-cell subset was previously shown to be necessary for development of the disease. Thus, these findings may be important for understanding the pathogenesis of autoimmune diabetes mellitus and potentially also for that of other T-cell-mediated autoimmune diseases.

Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes.

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

Transcription factor CTF1 acts as a chromatin domain boundary that shields human telomeric genes from silencing.

Telomeres are associated with chromatin-mediated silencing of genes in their vicinity. However, how epigenetic markers mediate mammalian telomeric silencing and whether specific proteins may counteract this effect are not known. We evaluated the ability of CTF1, a DNA- and histone-binding transcription factor, to prevent transgene silencing at human telomeres. CTF1 was found to protect a gene from silencing when its DNA-binding sites were interposed between the gene and the telomeric extremity, while it did not affect a gene adjacent to the telomere. Protein fusions containing the CTF1 histone-binding domain displayed similar activities, while mutants impaired in their ability to interact with the histone did not. Chromatin immunoprecipitation indicated the propagation of a hypoacetylated histone structure to various extents depending on the telomere. The CTF1 fusion protein was found to recruit the H2A.Z histone variant at the telomeric locus and to restore high histone acetylation levels to the insulated telomeric transgene. Histone lysine trimethylations were also increased on the insulated transgene, indicating that these modifications may mediate expression rather than silencing at human telomeres. Overall, these results indicate that transcription factors can act to delimit chromatin domain boundaries at mammalian telomeres, thereby blocking the propagation of a silent chromatin structure.