Philippe Demougin

The conserved transcriptome in human and rodent male gametogenesis

We report a cross-species expression profiling analysis of the human, mouse, and rat male meiotic transcriptional program, using enriched germ cell populations, whole gonads, and high-density oligonucleotide microarrays (GeneChips). Among 35% of the protein-coding genes present in rodent and human genomes that were found to be differentially expressed between germ cells and somatic controls, a key group of 357 conserved core loci was identified that displays highly similar meiotic and postmeiotic patterns of transcriptional induction across all three species. Genes known to be important for sexual reproduction are significantly enriched among differentially expressed core loci and a smaller group of conserved genes not detected in 17 nontesticular somatic tissues, correlating transcriptional activation and essential function in the male germ line. Some genes implicated in the etiology of cancer are found to be strongly transcribed in testis, suggesting that these genes may play unexpected roles in sexual reproduction. Expression profiling data further identified numerous conserved genes of biological and clinical interest previously unassociated with the mammalian male germ line.

Three-Dimensional Culture of Melanoma Cells Profoundly Affects Gene Expression Profile: A High Density Oligonucleotide Array Study

Growth in three‐dimensional (3D) architectures has been suggested to play an important role in tumor expansion and in the resistance of cancers to treatment with drugs or cytokines or irradiation. To obtain an insight into underlying molecular mechanisms, we addressed gene expression profiles of NA8 melanoma cells cultured in bidimensional monolayers (2D) or in 3D multicellular tumor spheroids (MCTS). MCTS containing 10‐30,000 cells were generated upon overnight culture in poly‐Hydroxyethylmethacrylate (polyHEMA) coated plates. Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long‐term culture (>10 days), however, MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with ≥threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, ≥threefold, upregulation of the expression of >100 transcripts while 73 were ≥threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO‐α, ‐β, and γ), IL‐8, CCL20 (MIP‐3α), and Angiopoietin‐like 4 were significantly upregulated, whereas basic FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real‐time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process.

Epigenetic Modification of the Glucocorticoid Receptor Gene Is Linked to Traumatic Memory and Post-Traumatic Stress Disorder Risk in Genocide Survivors

Recent evidence suggests that altered expression and epigenetic modification of the glucocorticoid receptor gene (NR3C1) are related to the risk of post-traumatic stress disorder (PTSD). The underlying mechanisms, however, remain unknown. Because glucocorticoid receptor signaling is known to regulate emotional memory processes, particularly in men, epigenetic modifications of NR3C1 might affect the strength of traumatic memories. Here, we found that increased DNA methylation at the NGFI-A (nerve growth factor-induced protein A) binding site of the NR3C1 promoter was associated with less intrusive memory of the traumatic event and reduced PTSD risk in male, but not female survivors of the Rwandan genocide. NR3C1 methylation was not significantly related to hyperarousal or avoidance symptoms. We further investigated the relationship between NR3C1 methylation and memory functions in a neuroimaging study in healthy subjects. Increased NR3C1 methylation–which was associated with lower NR3C1 expression–was related to reduced picture recognition in male, but not female subjects. Furthermore, we found methylation-dependent differences in recognition memory-related brain activity in men. Together, these findings indicate that an epigenetic modification of the glucocorticoid receptor gene promoter is linked to interindividual and gender-specific differences in memory functions and PTSD risk.

PKCα is genetically linked to memory capacity in healthy subjects and to risk for posttraumatic stress disorder in genocide survivors

Strong memory of a traumatic event is thought to contribute to the development and symptoms of posttraumatic stress disorder (PTSD). Therefore, a genetic predisposition to build strong memories could lead to increased risk for PTSD after a traumatic event. Here we show that genetic variability of the gene encoding PKCα (PRKCA) was associated with memory capacity—including aversive memory—in nontraumatized subjects of European descent. This finding was replicated in an independent sample of nontraumatized subjects, who additionally underwent functional magnetic resonance imaging (fMRI). fMRI analysis revealed PRKCA genotype-dependent brain activation differences during successful encoding of aversive information. Further, the identified genetic variant was also related to traumatic memory and to the risk for PTSD in heavily traumatized survivors of the Rwandan genocide. Our results indicate a role for PKCα in memory and suggest a genetic link between memory and the risk for PTSD.

Forgetting Is Regulated via Musashi-Mediated Translational Control of the Arp2/3 Complex

A plastic nervous system requires the ability not only to acquire and store but also to forget. Here, we report that musashi (msi-1) is necessary for time-dependent memory loss in C. elegans. Tissue-specific rescue demonstrates that MSI-1 function is necessary in the AVA interneuron. Using RNA-binding protein immunoprecipitation (IP), we found that MSI-1 binds to mRNAs of three subunits of the Arp2/3 actin branching regulator complex in vivo and downregulates ARX-1, ARX-2, and ARX-3 translation upon associative learning. The role of msi-1 in forgetting is also reflected by the persistence of learning-induced GLR-1 synaptic size increase in msi-1 mutants. We demonstrate that memory length is regulated cooperatively through the activation of adducin (add-1) and by the inhibitory effect of msi-1. Thus, a GLR-1/MSI-1/Arp2/3 pathway induces forgetting and represents a novel mechanism of memory decay by linking translational control to the structure of the actin cytoskeleton in neurons.

Global human tissue profiling and protein network analysis reveals distinct levels of transcriptional germline-specificity and identifies target genes for male infertility

BACKGROUND Mammalian spermatogenesis is a process that involves a complex expression program in both somatic and germ cells present in the male gonad. A number of studies have attempted to define the transcriptome of male meiosis and gametogenesis in rodents and primates. Few human transcripts, however, have been associated with testicular somatic cells and germ cells at different post-natal developmental stages and little is known about their level of germline-specificity compared with non-testicular tissues. METHODS We quantified human transcripts using GeneChips and a total of 47 biopsies from prepubertal children diagnosed with undescended testis, infertile adult patients whose spermatogenesis is arrested at consecutive stages and fertile control individuals. These results were integrated with data from enriched normal germ cells, non-testicular expression data, phenotype information, predicted regulatory DNA-binding motifs and interactome data. RESULTS Among 3580 genes for which we found differential transcript concentrations in somatic and germ cells present in human testis, 933 were undetectable in 45 embryonic and adult non-testicular tissues, including many that were corroborated at protein level by published gene annotation data and histological high-throughput protein immunodetection assays. Using motif enrichment analyses, we identified regulatory promoter elements likely involved in germline development. Finally, we constructed a regulatory disease network for human fertility by integrating expression signals, interactome information, phenotypes and functional annotation data. CONCLUSIONS Our results provide broad insight into the post-natal human testicular transcriptome at the level of cell populations and in a global somatic tissular context. Furthermore, they yield clues for genetic causes of male infertility and will facilitate the identification of novel cancer/testis genes as targets for cancer immunotherapies.

NPR1 Kinase and RSP5-BUL1/2 Ubiquitin Ligase Control GLN3-dependent Transcription in Saccharomyces cerevisiae

The GATA transcription factors GLN3 and GAT1 activate nitrogen-regulated genes in Saccharomyces cerevisiae. NPR1 is a protein kinase that controls post-Golgi sorting of amino acid permeases. In the presence of a good nitrogen source, TOR (target of rapamycin) maintains GLN3 and NPR1 phosphorylated and inactive by inhibiting the type 2A-related phosphatase SIT4. We identified NPR1 as a regulator of GLN3. Specifically, loss of NPR1 causes nuclear translocation and activation of GLN3, but not GAT1, in nitrogen-rich conditions. NPR1-mediated inhibition of GLN3 is independent of the phosphatase SIT4. We also demonstrate that the E3/E4 ubiquitin-protein ligase proteins RSP5 and BUL1/2 are required for GLN3 activation under poor nitrogen conditions. Thus, NPR1 and BUL1/2 antagonistically control GLN3-dependent transcription, suggesting a role for regulated ubiquitination in the control of nutrient-responsive transcription.

CPEB3 is associated with human episodic memory

Cytoplasmic polyadenylation element-binding (CPEB) proteins are crucial for synaptic plasticity and memory in model organisms. A highly conserved, mammalian-specific short intronic sequence within CPEB3 has been identified as a ribozyme with self-cleavage properties. In humans, the ribozyme sequence is polymorphic and harbors a single nucleotide polymorphism that influences cleavage activity of the ribozyme. Here we show that this variation is related to performance in an episodic memory task and that the effect of the variation depends on the emotional valence of the presented material. Our data suggest a role for human CPEB3 in human episodic memory.

A peripheral epigenetic signature of immune system genes is linked to neocortical thickness and memory

Increasing age is tightly linked to decreased thickness of the human neocortex. The biological mechanisms that mediate this effect are hitherto unknown. The DNA methylome, as part of the epigenome, contributes significantly to age-related phenotypic changes. Here, we identify an epigenetic signature that is associated with cortical thickness (P=3.86 × 10−8) and memory performance in 533 healthy young adults. The epigenetic effect on cortical thickness was replicated in a sample comprising 596 participants with major depressive disorder and healthy controls. The epigenetic signature mediates partially the effect of age on cortical thickness (P<0.001). A multilocus genetic score reflecting genetic variability of this signature is associated with memory performance (P=0.0003) in 3,346 young and elderly healthy adults. The genomic location of the contributing methylation sites points to the involvement of specific immune system genes. The decomposition of blood methylome-wide patterns bears considerable potential for the study of brain-related traits.

A genome-wide survey of human short-term memory

Recent advances in the development of high-throughput genotyping platforms allow for the unbiased identification of genes and genomic sequences related to heritable traits. In this study, we analyzed human short-term memory, which refers to the ability to remember information over a brief period of time and which has been found disturbed in many neuropsychiatric conditions, including schizophrenia and depression. We performed a genome-wide survey at 909 622 polymorphic loci and report six genetic variations significantly associated with human short-term memory performance after genome-wide correction for multiple comparisons. A polymorphism within SCN1A (encoding the α subunit of the type I voltage-gated sodium channel) was replicated in three independent populations of 1699 individuals. Functional magnetic resonance imaging during an n-back working memory task detected SCN1A allele-dependent activation differences in brain regions typically involved in working memory processes. These results suggest an important role for SCN1A in human short-term memory.