Jean Mosser

Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1 , and Atoh8 in the mouse liver

Although hepcidin expression was shown to be induced by the BMP/Smad signaling pathway, it is not yet known how iron regulates this pathway and what its exact molecular targets are. We therefore assessed genome-wide liver transcription profiles of mice of 2 genetic backgrounds fed iron-deficient, -balanced, or -enriched diets. Among 1419 transcripts significantly modulated by the dietary iron content, 4 were regulated similarly to the hepcidin genes Hamp1 and Hamp2. They are coding for Bmp6, Smad7, Id1, and Atoh8 all related to the Bmp/Smad pathway. As shown by Western blot analysis, variations in Bmp6 expression induced by the diet iron content have for functional consequence similar changes in Smad1/5/8 phosphorylation that leads to formation of heteromeric complexes with Smad4 and their translocation to the nucleus. Gene expression variations induced by secondary iron deficiency or iron overload were compared with those consecutive to Smad4 and Hamp1 deficiency. Iron overload developed by Smad4- and Hamp1-deficient mice also increased Bmp6 transcription. However, as shown by analysis of mice with liver-specific disruption of Smad4, activation of Smad7, Id1, and Atoh8 transcription by iron requires Smad4. This study points out molecules that appear to play a critical role in the control of systemic iron balance.

DNA methylation in glioblastoma: impact on gene expression and clinical outcome

BackgroundChanges in promoter DNA methylation pattern of genes involved in key biological pathways have been reported in glioblastoma. Genome-wide assessments of DNA methylation levels are now required to decipher the epigenetic events involved in the aggressive phenotype of glioblastoma, and to guide new treatment strategies.ResultsWe performed a whole-genome integrative analysis of methylation and gene expression profiles in 40 newly diagnosed glioblastoma patients. We also screened for associations between the level of methylation of CpG sites and overall survival in a cohort of 50 patients uniformly treated by surgery, radiotherapy and chemotherapy with concomitant and adjuvant temozolomide (STUPP protocol). The methylation analysis identified 616 CpG sites differentially methylated between glioblastoma and control brain, a quarter of which was differentially expressed in a concordant way. Thirteen of the genes with concordant CpG sites displayed an inverse correlation between promoter methylation and expression level in glioblastomas: B3GNT5, FABP7, ZNF217, BST2, OAS1, SLC13A5, GSTM5, ME1, UBXD3, TSPYL5, FAAH, C7orf13, and C3orf14. Survival analysis identified six CpG sites associated with overall survival. SOX10 promoter methylation status (two CpG sites) stratified patients similarly to MGMT status, but with a higher Area Under the Curve (0.78 vs. 0.71, p- value < 5e-04). The methylation status of the FNDC3B, TBX3, DGKI, and FSD1 promoters identified patients with MGMT-methylated tumors that did not respond to STUPP treatment (p- value < 1e-04).ConclusionsThis study provides the first genome-wide integrative analysis of DNA methylation and gene expression profiles obtained from the same GBM cohort. We also present a methylome-based survival analysis for one of the largest uniformly treated GBM cohort ever studied, for more than 27,000 CpG sites. We have identified genes whose expression may be tightly regulated by epigenetic mechanisms and markers that may guide treatment decisions.

Common Variants in the BMP2, BMP4, and HJV Genes of the Hepcidin Regulation Pathway Modulate HFE Hemochromatosis Penetrance

Most cases of genetic hemochromatosis (GH) are associated with the HFE C282Y/C282Y (p.Cys282Tyr/p.Cys282Tyr) genotype in white populations. The symptoms expressed by C282Y homozygotes are extremely variable. Only a few suffer from an overt disease. Several studies have suggested that, in addition to environmental factors, a genetic component could explain a substantial part of this phenotypic variation, although very few genetic factors have been identified so far. In the present study, we tested the association between common variants in candidate genes and hemochromatosis penetrance, in a large sample of C282Y homozygotes, using pretherapeutic serum ferritin level as marker of hemochromatosis penetrance. We focused on two biologically relevant gene categories: genes involved in non-HFE GH (TFR2, HAMP, and SLC40A1) and genes involved in the regulation of hepcidin expression, including genes from the bone morphogenetic protein (BMP) regulatory pathway (BMP2, BMP4, HJV, SMAD1, SMAD4, and SMAD5) and the IL6 gene from the inflammation-mediated regulation pathway. A significant association was detected between serum ferritin level and rs235756, a common single-nucleotide polymorphism (SNP) in the BMP2 genic region (P=4.42x10-5). Mean ferritin level, adjusted for age and sex, is 655 ng/ml among TT genotypes, 516 ng/ml in TC genotypes, and 349 ng/ml in CC genotypes. Our results further suggest an interactive effect on serum ferritin level of rs235756 in BMP2 and a SNP in HJV, with a small additive effect of a SNP in BMP4. This first reported association between common variants in the BMP pathway and iron burden suggests that full expression of HFE hemochromatosis is linked to abnormal liver expression of hepcidin, not only through impairment in the HFE function but also through functional modulation in the BMP pathway. Our results also highlight the BMP regulation pathway as a good candidate for identification of new modifier genes.

Orchestrated transcription of biological processes in the marine picoeukaryote Ostreococcus exposed to light/dark cycles.

BackgroundPicoeukaryotes represent an important, yet poorly characterized component of marine phytoplankton. The recent genome availability for two species of Ostreococcus and Micromonas has led to the emergence of picophytoplankton comparative genomics. Sequencing has revealed many unexpected features about genome structure and led to several hypotheses on Ostreococcus biology and physiology. Despite the accumulation of genomic data, little is known about gene expression in eukaryotic picophytoplankton.ResultsWe have conducted a genome-wide analysis of gene expression in Ostreococcus tauri cells exposed to light/dark cycles (L/D). A Bayesian Fourier Clustering method was implemented to cluster rhythmic genes according to their expression waveform. In a single L/D condition nearly all expressed genes displayed rhythmic patterns of expression. Clusters of genes were associated with the main biological processes such as transcription in the nucleus and the organelles, photosynthesis, DNA replication and mitosis.ConclusionsLight/Dark time-dependent transcription of the genes involved in the main steps leading to protein synthesis (transcription basic machinery, ribosome biogenesis, translation and aminoacid synthesis) was observed, to an unprecedented extent in eukaryotes, suggesting a major input of transcriptional regulations in Ostreococcus. We propose that the diurnal co-regulation of genes involved in photoprotection, defence against oxidative stress and DNA repair might be an efficient mechanism, which protects cells against photo-damage thereby, contributing to the ability of O. tauri to grow under a wide range of light intensities.

NOD2/CARD15 gene polymorphisms in Crohn's disease: a genotype-phenotype analysis

Objectives Three recently identified NOD2/CARD15 mutations have been described associated with an increased susceptibility Crohns disease (CD). Our aim was to examine the potential association of these NOD2 mutations with CD and different subsets of CD phenotypes in our population. Methods Two hundred and five well-defined CD patients from north-western France and 95 ethnically matched healthy controls were genotyped for mutations R702W, G908R and Leu1007insC by DNA sequencing. Allele and genotype frequencies of NOD2 variants were examined in the whole series of CD and in different subgroups of CD phenotypes defined by the clinical characteristics of the Vienna classification (age at diagnosis, location and behaviour) or by histological features (granuloma). Results Carriers of at least one NOD2/CARD15 variant were significantly more frequent in CD than in controls (38.0% versus 20.0%, P < 0.002), and the R702W allele was the most significant contributor to this NOD2 association with CD. Homozygotes and compound heterozygotes combined had a higher risk of CD (odds ratio = 12.0, P < 0.0026) than simple heterozygotes for any variant (odds ratio = 2.2, P < 0.013) compared with subjects with no variant. Univariate analysis revealed that carriage of at least one NOD2 mutation was significantly associated with ileal involvement (P < 0.03), and stricturing evolution (P < 0.0015). Granuloma was associated with an excess of the R702W allele (16.1% versus 8.0%, Pc < 0.035), and was correlated with a young age at diagnosis, whatever the NOD2/CARD15 genotype. Multivariate analysis demonstrated that carriage of NOD2/CARD15 mutants, especially R702W, was primarily and independently associated both with stricturing evolution of CD and the presence of granuloma. Conclusions In our population, all NOD2/CARD15 mutant genotypes, especially compound heterozygosity, were found to increase the risk of CD, but R702W was the sole allele showing a significant association with CD. In addition, we confirm the positive and independent association of the R702W mutation with stricturing behaviour and describe a second one with the presence of granuloma.

Gender‐specific phenotypic expression and screening strategies in C282Y‐linked haemochromatosis: a study of 9396 French people

Summary. Most features of C282Y‐linked haemochromatosis support the implementation of population screening of the disorder in Caucasians. However, the penetrance of C282Y homozygosity is poorly documented and the strategy for population screening remains debated. Nine thousand three hundred and ninety‐six subjects (3367 men, aged 25–40 years, and 6029 women, aged 35–50 years), attending three Health Appraisal Centres, were genotyped and assessed with respect to clinical and biochemical signs of haemochromatosis. Discriminant, logistic regression and graphic analysis were used to predict homozygosity. Results were validated in 135 homozygotes detected through other family and population studies. Fifty‐four subjects (10 men and 44 women) were homozygous for C282Y. All men had abnormal iron status and most had mild clinical symptoms compatible with haemochromatosis. Identification of all homozygous men required a transferrin saturation (TS) threshold of 50% in the study group (90% specificity) and of 40% in the validation group. Homozygous women differed clinically from non‐homozygotes for the presence of distal arthralgias only (18%vs 6%, P < 0·03). Thirteen (29%) were iron‐deficient (serum ferritin < 13 µg/l) and undetectable by biochemical tests. Although the population studied was not fully representative of the general population, our data strongly suggests that, in young men, large‐scale screening for C282Y homozygosity is justified and can be achieved by using TS prescreening. However, in premenopausal women, large‐scale screening remains to be justified with respect to the natural history of haemochromatosis and should be directly genotypic.

The protein coded by the X-adrenoleukodystrophy gene is a peroxisomal integral membrane protein.

The gene for adrenoleukodystrophy (X‐ALD), a peroxisomal disease characterized by excessive accumulation of very long‐chain (VLC) fatty acids (> C22;0), has recently been identified by positional cloning, and it is predicted to encode a protein (ALD‐P) of 745 amino acids [(1993) Nature 361, 726]. Using Western blot analysis of subcellular organelles purified by isopycnic density gradient centrifugation from X‐ALD and control fibroblasts, we show that the monoclonal antibodies directed against ALD‐P cross‐react with a 75 kDa protein in intact peroxisomes and that ALD‐P is an integral component of the peroxisomal membrane. Moreover, no signal for ALD‐P was detected in peroxisomes from X‐ALD patients with deletion of the ALD gene.

Light-Dependent Regulation of Cell Division in Ostreococcus: Evidence for a Major Transcriptional Input

Cell division often occurs at specific times of the day in animal and photosynthetic organisms. Studies in unicellular photosynthetic algae, such as Chlamydomonas or Euglena, have shown that the photoperiodic control of cell division is mediated through the circadian clock. However, the underlying mechanisms remain unknown. We have studied the molecular basis of light-dependent control of cell division in the unicellular green alga Ostreococcus. We found that cell division obeys a circadian oscillator in Ostreococcus. We provide evidence suggesting that the clock may, at least in part, regulate directly cell division independently of the metabolism. Combined microarray and quantitative real-time reverse transcription-polymerase chain reaction analysis of the main core cell cycle gene expression revealed an extensive transcriptional regulation of cell division by the photoperiod in Ostreococcus. Finally, transcription of the main core cell cycle genes, including cyclins and cyclin-dependent kinases, was shown to be under circadian control in Ostreococcus, suggesting that these genes are potential targets of the circadian clock in the control of cell division.

Integrative genome‐wide analysis reveals a robust genomic glioblastoma signature associated with copy number driving changes in gene expression

Glioblastoma multiforme shows multiple chromosomal aberrations, the impact of which on gene expression remains unclear. To investigate this relationship and to identify putative initiating genomic events, we integrated a paired copy number and gene expression survey in glioblastoma using whole human genome arrays. Loci of recurrent copy number alterations were combined with gene expression profiles obtained on the same tumor samples. We identified a set of 406 “cis‐acting DNA targeted genes” corresponding to genomic aberrations with direct copy‐number‐driving changes in gene expression, defined as genes with either significantly concordant or correlated changes in DNA copy number and expression. Functional annotation revealed that these genes participate in key processes of cancer cell biology, providing insights into the genetic mechanisms driving glioblastoma. The robustness of the gene selection was validated on an external microarray data set including 81 glioblastomas and 23 non‐neoplastic brain samples. The integration of array CGH and gene expression data highlights a robust cis‐acting DNA targeted genes signature that may be critical for glioblastoma progression, with two tumor suppressor genes PCDH9 and STARD13 that could be involved in tumor invasiveness and resistance to etoposide.

Distinct effects of human glioblastoma immunoregulatory molecules programmed cell death ligand-1 (PDL-1) and indoleamine 2,3-dioxygenase (IDO) on tumour-specific T cell functions.

Immunotherapy is a promising new treatment for patients suffering from glioma, in particular glioblastoma multiforme (GBM). However, tumour cells use different mechanisms to escape the immune responses induced by the treatment. As many other tumours, gliomas express or secrete several immunosuppressive molecules that regulate immune cell functions. In this study, we first analysed FasL, HLA-G, IDO, PDL-1 and TGF-beta1, -beta2 and -beta3 expression by transcriptomic microarray analysis in a series of 20 GBM samples and found respectively 15%, 60%, 85%, 30%, 70%, 80% and 35% of positive specimens. mRNA expression was then confirmed in 10 GBM primary cell lines and 2 immortalised cell lines U251 and U87MG. Furthermore, the protein expression of PDL-1, IDO activity and TGF-beta2 secretion were found on most of the untreated GBM primary cell lines. Remarkably, treatment with IFN-gamma increased the PDL-1 cell surface expression and the IDO activity, but reduced the TGF-beta2 secretion of GBM cell lines. We finally analysed the immunosuppressive effects of IDO, PDL-1 and TGF-beta1-3 by measuring IFN-gamma production and cell cytotoxicity activity of tumour antigen-specific T cells. PDL-1 partially affected the IFN-gamma production of antigen-specific T cells in response to GBM primary cell lines, and IDO inhibited lymphocyte proliferation induced by lectins. None of these molecules directly affected the T cell cytotoxicity function. Due to the functional role of PDL-1 and IDO molecules expressed by GBM cells, one could expect that blocking these molecules in the immunotherapy strategies would reinforce the efficiency of these treatments of GBM patients.