André Nantel

Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

A Human-Curated Annotation of the Candida albicans Genome

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.

Biofilm Matrix Regulation by Candida albicans Zap1

The zinc-responsive transcription factor Zap1 has a striking role in fungal biofilm formation and is reported to regulate matrix formation.

Molecular Profiling Identifies Prognostic Subgroups of Pediatric Glioblastoma and Shows Increased YB-1 Expression in Tumors

PURPOSE Pediatric glioblastoma (pGBM) is a rare, but devastating brain tumor. In contrast to GBM in adults (aGBM), little is known about the mechanisms underlying its development. Our aim is to gain insight into the molecular pathways of pGBM. MATERIALS AND METHODS Thirty-two pGBM and seven aGBM samples were investigated using biochemical and transcriptional profiling. Ras and Akt pathway activation was assessed through the phosphorylation of downstream effectors, and gene expression profiles were generated using the University Health Network Human 19K cDNA arrays. Results were validated using real-time polymerase chain reaction and immunohistochemistry and compared with existing data sets on aGBM. RESULTS There are at least two subsets of pGBM. One subset, associated with Ras and Akt pathway activation, has very poor prognosis and exhibits increased expression of genes related to proliferation and to a neural stem-cell phenotype, similar to findings in aggressive aGBM. This subset was still molecularly distinguishable from aGBM after unsupervised and supervised analysis of expression profiles. A second subset, with better prognosis, is not associated with activation of Akt and Ras pathways, may originate from astroglial progenitors, and does not express gene signatures and markers shown to be associated with long-term survival in aGBM. Both subsets of pGBM show overexpression of Y-box-protein-1 that may help drive oncogenesis in this tumor. CONCLUSION Our work, the first study of gene expression profiles in pGBM, provides valuable insight into active pathways and targets in a cancer with minimal survival, and suggests that these tumors cannot be understood exclusively through studies of aGBM.

Genomics of hybrid poplar (Populus trichocarpa× deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full-length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect-induced defences in poplar

As part of a genomics strategy to characterize inducible defences against insect herbivory in poplar, we developed a comprehensive suite of functional genomics resources including cDNA libraries, expressed sequence tags (ESTs) and a cDNA microarray platform. These resources are designed to complement the existing poplar genome sequence and poplar (Populus spp.) ESTs by focusing on herbivore‐ and elicitor‐treated tissues and incorporating normalization methods to capture rare transcripts. From a set of 15 standard, normalized or full‐length cDNA libraries, we generated 139 007 3′‐ or 5′‐end sequenced ESTs, representing more than one‐third of the c. 385 000 publicly available Populus ESTs. Clustering and assembly of 107 519 3′‐end ESTs resulted in 14 451 contigs and 20 560 singletons, altogether representing 35 011 putative unique transcripts, or potentially more than three‐quarters of the predicted c. 45 000 genes in the poplar genome. Using this EST resource, we developed a cDNA microarray containing 15 496 unique genes, which was utilized to monitor gene expression in poplar leaves in response to herbivory by forest tent caterpillars (Malacosoma disstria). After 24 h of feeding, 1191 genes were classified as up‐regulated, compared to only 537 down‐regulated. Functional classification of this induced gene set revealed genes with roles in plant defence (e.g. endochitinases, Kunitz protease inhibitors), octadecanoid and ethylene signalling (e.g. lipoxygenase, allene oxide synthase, 1‐aminocyclopropane‐1‐carboxylate oxidase), transport (e.g. ABC proteins, calreticulin), secondary metabolism [e.g. polyphenol oxidase, isoflavone reductase, (–)‐germacrene D synthase] and transcriptional regulation [e.g. leucine‐rich repeat transmembrane kinase, several transcription factor classes (zinc finger C3H type, AP2/EREBP, WRKY, bHLH)]. This study provides the first genome‐scale approach to characterize insect‐induced defences in a woody perennial providing a solid platform for functional investigation of plant–insect interactions in poplar.

Transcriptional rewiring of fungal galactose-metabolism circuitry.

BACKGROUND The Leloir-pathway genes encode the enzymatic machinery involved in the metabolism of galactose. RESULTS In the distantly related fungi Saccharomyces cerevisiae and Candida albicans, the genes encoding these enzymes are syntenically arranged, but the upstream regulatory regions are highly divergent. In S. cerevisiae, the Leloir-pathway genes are positively regulated by Gal4p acting through the UAS(G) sequence CGG(N(11))CCG. However, in C. albicans, the Gal4p and UAS(G) combination is found to regulate genes unrelated to galactose metabolism. We identified a palindromic sequence that acts to control GAL10 expression in C. albicans in the presence of galactose. This palindrome is found upstream of other Leloir-pathway genes in C. albicans, and in the absence of other regulatory sequences, activation of expression through this sequence in the presence of galactose requires Cph1p, the homolog of the Ste12p transcription factor of S. cerevisiae. CONCLUSIONS Although the cellular process of galactose induction of the Leloir pathway is conserved between the two organisms, the regulatory circuits achieving the cellular process are completely distinct.

Population genomics of drug resistance in Candida albicans

We followed adaptation in experimental microbial populations to inhibitory concentrations of an antimicrobial drug. The evolution of drug resistance was accompanied in all cases by changes in gene expression that persisted in the absence of the drug; the new patterns of gene expression were constitutive. The changes in gene expression occurred in four replicate populations of the pathogenic fungus Candida albicans during 330 generations of evolution in the presence of the antifungal drug fluconazole. Genome-wide expression profiling of over 5,000 ORFs identified 301 whose expression was significantly modulated. Cluster analysis identified three distinct patterns of gene expression underlying adaptation to the drug. One pattern was unique to one population and included up-regulation of the multidrug ATP-binding cassette transporter gene, CDR2. A second pattern occurred at a late stage of adaptation in three populations; for two of these populations profiled earlier in their evolution, a different pattern was observed at an early stage of adaptation. The succession of early- and late-stage patterns of gene expression, both of which include up-regulation of the multidrug major facilitator transporter gene, MDR1, must represent a common program of adaptation to this antifungal drug. The three patterns of gene expression were also identified in fluconazole-resistant clinical isolates, providing further evidence that these patterns represent common programs of adaptation to fluconazole.

Assembly of the Candida albicans genome into sixteen supercontigs aligned on the eight chromosomes

BackgroundThe 10.9× genomic sequence of Candida albicans, the most important human fungal pathogen, was published in 2004. Assembly 19 consisted of 412 supercontigs, of which 266 were a haploid set, since this fungus is diploid and contains an extensive degree of heterozygosity but lacks a complete sexual cycle. However, sequences of specific chromosomes were not determined.ResultsSupercontigs from Assembly 19 (183, representing 98.4% of the sequence) were assigned to individual chromosomes purified by pulse-field gel electrophoresis and hybridized to DNA microarrays. Nine Assembly 19 supercontigs were found to contain markers from two different chromosomes. Assembly 21 contains the sequence of each of the eight chromosomes and was determined using a synteny analysis with preliminary versions of the Candida dubliniensis genome assembly, bioinformatics, a sequence tagged site (STS) map of overlapping fosmid clones, and an optical map. The orientation and order of the contigs on each chromosome, repeat regions too large to be covered by a sequence run, such as the ribosomal DNA cluster and the major repeat sequence, and telomere placement were determined using the STS map. Sequence gaps were closed by PCR and sequencing of the products. The overall assembly was compared to an optical map; this identified some misassembled contigs and gave a size estimate for each chromosome.ConclusionAssembly 21 reveals an ancient chromosome fusion, a number of small internal duplications followed by inversions, and a subtelomeric arrangement, including a new gene family, the TLO genes. Correlations of position with relatedness of gene families imply a novel method of dispersion. The sequence of the individual chromosomes of C. albicans raises interesting biological questions about gene family creation and dispersion, subtelomere organization, and chromosome evolution.

Transcriptional Regulation of Carbohydrate Metabolism in the Human Pathogen Candida albicans

Glycolysis is a metabolic pathway that is central to the assimilation of carbon for either respiration or fermentation and therefore is critical for the growth of all organisms. Consequently, glycolytic transcriptional regulation is important for the metabolic flexibility of pathogens in their attempts to colonize diverse niches. We investigated the transcriptional control of carbohydrate metabolism in the human fungal pathogen Candida albicans and identified two factors, Tye7p and Gal4p, as key regulators of glycolysis. When respiration was inhibited or oxygen was limited, a gal4tye7 C. albicans strain showed a severe growth defect when cultured on glucose, fructose or mannose as carbon sources. The gal4tye7 strain displayed attenuated virulence in both Galleria and mouse models as well, supporting the connection between pathogenicity and metabolism. Chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP) and transcription profiling revealed that Tye7p bound the promoter sequences of the glycolytic genes and activated their expression during growth on either fermentable or non-fermentable carbon sources. Gal4p also bound the glycolytic promoter sequences and activated the genes although to a lesser extent than Tye7p. Intriguingly, binding and activation by Gal4p was carbon source-dependent and much stronger during growth on media containing fermentable sugars than on glycerol. Furthermore, Tye7p and Gal4p were responsible for the complete induction of the glycolytic genes under hypoxic growth conditions. Tye7p and Gal4p also regulated unique sets of carbohydrate metabolic genes; Tye7p bound and activated genes involved in trehalose, glycogen, and glycerol metabolism, while Gal4p regulated the pyruvate dehydrogenase complex. This suggests that Tye7p represents the key transcriptional regulator of carbohydrate metabolism in C. albicans and Gal4p provides a carbon source-dependent fine-tuning of gene expression while regulating the metabolic flux between respiration and fermentation pathways.

Pyruvate kinase deficiency in mice protects against malaria

The global health impact of malaria is enormous, with an estimated 300–500 million clinical cases and 1 million annual deaths. In humans, initial susceptibility to infection with Plasmodium species, disease severity and ultimate outcome of malaria (self-healing or lethal) are under complex genetic control. Alleles associated with sickle cell anemia, β-thalassemia and deficiency in glucose-6-phosphate dehydrogenase have a protective effect against malaria and may have been retained by positive selection in areas of endemic malaria. Likewise, genetic variations in erythrocyte antigens and levels of host cytokines affect type and severity of disease. A mouse model of infection with Plasmodium chabaudi was used to study the genetic component of malaria susceptibility. Segregation analyses in informative F2 crosses derived from resistant C57BL/6J and susceptible A/J, C3H and SJL strains using extent of blood stage replication of the parasite and survival as traits mapped three P. chabaudi resistance (Char) loci on chromosomes 9 (Char1), 8 (Char2) and 17 (Char3, MHC-linked). Recombinant congenic strains AcB55 and AcB61 are unusually resistant to malaria despite carrying susceptibility alleles at Char1 and Char2. Malaria resistance in AcB55 and AcB61 is associated with splenomegaly and constitutive reticulocytosis, is inherited in an autosomal recessive fashion and is controlled by a locus on chromosome 3 (Char4). Sequencing of candidate genes from the Char4 region identified a loss-of-function mutation (269T→A, resulting in the amino acid substitution I90N) in the pyruvate kinase gene (Pklr) that underlies the malaria resistance in AcB55 and AcB61. These results suggest that pyruvate kinase deficiency may similarly protect humans against malaria.