Extent and patterns of MGMT promoter methylation in glioblastoma- and respective glioblastoma-derived spheres.

Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity. Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255-66. (C)2010 AACR.

Deviant trajectories of cortical maturation in 22q11.2 deletion syndrome (22q11DS): a cross-sectional and longitudinal study.

22q11.2 deletion syndrome (22q11DS) is associated with an increased susceptibility to develop schizophrenia. Despite a large body of literature documenting abnormal brain structure in 22q11DS, cerebral changes associated with brain maturation in 22q11DS remained largely unexplored. To map cortical maturation from childhood to adulthood in 22q11.2 deletion syndrome, we used cerebral MRI from 59 patients with 22q11DS, aged 6 to 40, and 80 typically developing controls; three year follow-up assessments were also available for 32 patients and 31 matched controls. Cross-sectional cortical thickness trajectories during childhood and adolescence were approximated in age bins. Repeated-measures were also conducted with the longitudinal data. Within the group of patients with 22q11DS, exploratory measures of cortical thickness differences related to COMT polymorphism, IQ, and schizophrenia were also conducted. We observed deviant trajectories of cortical thickness changes with age in patients with 22q11DS. In affected preadolescents, larger prefrontal thickness was observed compared to age-matched controls. Afterward, we observed greater cortical loss in 22q11DS with a convergence of cortical thickness values by the end of adolescence. No compelling evidence for an effect of COMT polymorphism on cortical maturation was observed. Within 22q11DS, significant differences in cortical thickness were related to cognitive level in children and adolescents, and to schizophrenia in adults. Deviant trajectories of cortical thickness from childhood to adulthood provide strong in vivo cues for a defect in the programmed synaptic elimination, which in turn may explain the susceptibility of patients with 22q11DS to develop psychosis.

Acetylcholine-induced vasodilation and reactive hyperemia are not affected by acute cyclo-oxygenase inhibition in human skin

Résumé Il est actuellement reconnu que l'endothélium vasculaire joue un rôle primordial dans la genèse des maladies cardiovasculaires, notamment l'artériosclérose. Dès lors, il est important de pouvoir investiguer la fonction endothéliale en clinique. Pour ce faire, il est particulièrement simple d'examiner la microcirculation cutanée, car celle-ci est très simplement accessible, de manière non-invasive, par fluxmétrie laser-Doppler. Pratiquement, on mesure l'augmentation du flux sanguin dermique en réponse à des stimuli connus pour agir via l'endothélium vasculaire. Les stimuli endothélium-dépendants les plus courants sont l'interruption temporaire du flux sanguin qui est suivie d'une hyperémie réactive, et l'administration transcutanée d'acétylcholine (Ach) par iontophorèse. La iontophorèse consiste à obtenir le transfert d' une substance ionisée, telle l'Ach, par l'application d'un courant électrique de polarité appropriée. L'objectif du présent travail était de déterminer le rôle des prostaglandines dans ces réponse vasodilatatrices dépendante de l'endothélium, rôle actuellement peu clair. 23 jeunes hommes volontaires non fumeurs et en bonne santé habituelle ont été examinés lors de deux visites séparées par 1 à 3 semaines. Lors de chaque visite, l'hyperémie réactive et la réponse vasodilatatrice à l'Ach ont été déterminées dans la peau de l'avant bras après administration soit d'un placebo, soit d'un inhibiteur de la cyclooxygénase (COX, enzyme qui contrôle la synthèse des prostaglandines). Chez certains sujets, l'inhibiteur était de l'acétylsalicylate de lysine (900 mg par voie intraveineuse). Chez d'autres sujets, il s'agissait d'indométhacine. (75 mg par voie orale). Comme la stimulation nociceptive liée au courant iontophorétique peut influencer la réponse à l'Ach, celle-ci a été déterminée en présence et en l'absence d'anesthésie de surface (crème de lidocaine). La réponse à l'Ach a été obtenue pour 4 doses différentes de cet agent (exprimées sous la forme de la densité de charge iontophorétique appliquée : 0.28, 1.4, 7, et 14 millicoulombs par cm2 de peau exposée). Le flux sanguin dermique était mesuré par imagerie laser-Doppler, une variante de la fluxmétrie laser-Doppler classique permettant l'exploration d'une surface de peau de taille arbitraire. Quelle que soit la condition testée, nous n'avons jamais observé la moindre influence de l'inhibition de la COX sur l'hyperémie réactive, ni sur la réponse à l'Ach. Cette dernière était augmentée significativement par l'anesthésie cutanée, que les sujets aient reçu ou non de l'acétylsalicylate de lysine ou de l'indométhacine . Par exemple, la réponses moyenne (±SD) à la plus haute dose d'Ach (testée sur 6 sujets, et exprimée en unités de perfusion, comme il est d'usage en fluxmétrie laser-Doppler ) était la suivante : en l'absence d'anesthésie : acétylsalicylate de lysine 339 ± 105, placebo 344 ± 68 ; avec l'anesthésie : acétylsalicylate de lysine 453 ± 76 , placebo 452 ± 65 (p * 0.001 pour les effets de l'anesthésie). En conclusion, nos résultats infirment une contribution des prostaglandines à l'hyperémie réactive ou à la vasodilatation induite par l'acétylcholine dans la microcirculation cutanée. Dans ce lit vasculaire, l'anesthésie locale accroît la vasodilatation induite par l'acétylcholine par un mécanisme indépendant des prostaglandines.

PPARs: nuclear receptors for fatty acids, eicosanoids, and xenobiotics.

The peroxisome proliferator-activated receptors have enjoyed the spotlight for many reasons. These transcription factors are ligand-inducible nuclear receptors that modulate gene expression in response to a broad spectrum of compounds. The recognition that PPARs are indeed nuclear receptors for polyunsaturated fatty acids, some eicosanoids and also lipid-lowering and antidiabetic drugs, has opened many exciting avenues of research and drug discovery. Recent studies on the PPAR function have extended the role of these transcription factors beyond energy homeostasis to master gene in adipogenesis and also determinants in inflammation control. While rapid advances have been made, it is clear that we are far from a global understanding of the mechanisms and functions of PPARs.

Extracellular histones in tissue injury and inflammation.

Neutrophil NETosis is an important element of host defense as it catapults chromatin out of the cell to trap bacteria, which then are killed, e.g., by the chromatin's histone component. Also, during sterile inflammation TNF-alpha and other mediators trigger NETosis, which elicits cytotoxic effects on host cells. The same mechanism should apply to other forms of regulated necrosis including pyroptosis, necroptosis, ferroptosis, and cyclophilin D-mediated regulated necrosis. Beyond these toxic effects, extracellular histones also trigger thrombus formation and innate immunity by activating Toll-like receptors and the NLRP3 inflammasome. Thereby, extracellular histones contribute to the microvascular complications of sepsis, major trauma, small vessel vasculitis as well as acute liver, kidney, brain, and lung injury. Finally, histones prevent the degradation of extracellular DNA, which promotes autoimmunization, anti-nuclear antibody formation, and autoimmunity in susceptible individuals. Here, we review the current evidence on the pathogenic role of extracellular histones in disease and discuss how to target extracellular histones to improve disease outcomes.

Extent and Patterns of MGMT Promoter Methylation in Glioblastoma- and Respective Glioblastoma-Derived Spheres.

PURPOSE: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. EXPERIMENTAL DESIGN: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. RESULTS: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity. CONCLUSIONS: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255-66. ©2010 AACR.

The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach.

DNA methylation is involved in a diversity of processes in bacteria, including maintenance of genome integrity and regulation of gene expression. Here, using Caulobacter crescentus as a model, we exploit genome-wide experimental methods to uncover the functions of CcrM, a DNA methyltransferase conserved in most Alphaproteobacteria. Using single molecule sequencing, we provide evidence that most CcrM target motifs (GANTC) switch from a fully methylated to a hemi-methylated state when they are replicated, and back to a fully methylated state at the onset of cell division. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. Strikingly, GANTC methylation is needed for the efficient transcription of dozens of genes that are essential for cell cycle progression, in particular for DNA metabolism and cell division. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteria.

A nucleosome-dependent static loop potentiates estrogen-regulated transcription from the Xenopus vitellogenin B1 promoter in vitro.

We describe the transcriptional potentiation in estrogen responsive transcription extracts of the Xenopus vitellogenin B1 gene promoter through the formation of a positioned nucleosome. Nuclease digestion and hydroxyl radical cleavage indicate that strong, DNA sequence-directed positioning of a nucleosome occurs between -300 and -140 relative to the start site of transcription. Deletion of this DNA sequence abolishes the potentiation of transcription due to nucleosome assembly. The wrapping of DNA around the histone core of the nucleosome positioned between -300 and -140 creates a static loop in which distal estrogen receptor binding sites are brought close to proximal promoter elements. This might facilitate interactions between the trans-acting factors themselves and/or RNA polymerase. Such a nucleosome provides an example of how chromatin structure might have a positive effect on the transcription process.

Large scale assessment of regulatory evolution and transcriptome complexity in mammals

AbstractIn addition to genetic changes affecting the function of gene products, changes in gene expression have been suggested to underlie many or even most of the phenotypic differences among mammals. However, detailed gene expression comparisons were, until recently, restricted to closely related species, owing to technological limitations. Thus, we took advantage of the latest technologies (RNA-Seq) to generate extensive qualitative and quantitative transcriptome data for a unique collection of somatic and germline tissues from representatives of all major mammalian lineages (placental mammals, marsupials and monotremes) and birds, the evolutionary outgroup.In the first major project of my thesis, we performed global comparative analyses of gene expression levels based on these data. Our analyses provided fundamental insights into the dynamics of transcriptome change during mammalian evolution (e.g., the rate of expression change across species, tissues and chromosomes) and allowed the exploration of the functional relevance and phenotypic implications of transcription changes at a genome-wide scale (e.g., we identified numerous potentially selectively driven expression switches).In a second project of my thesis, which was also based on the unique transcriptome data generated in the context of the first project we focused on the evolution of alternative splicing in mammals. Alternative splicing contributes to transcriptome complexity by generating several transcript isoforms from a single gene, which can, thus, perform various functions. To complete the global comparative analysis of gene expression changes, we explored patterns of alternative splicing evolution. This work uncovered several general and unexpected patterns of alternative splicing evolution (e.g., we found that alternative splicing evolves extremely rapidly) as well as a large number of conserved alternative isoforms that may be crucial for the functioning of mammalian organs.Finally, the third and final project of my PhD consisted in analyzing in detail the unique functional and evolutionary properties of the testis by exploring the extent of its transcriptome complexity. This organ was previously shown to evolve rapidly both at the phenotypic and molecular level, apparently because of the specific pressures that act on this organ and are associated with its reproductive function. Moreover, my analyses of the amniote tissue transcriptome data described above, revealed strikingly widespread transcriptional activity of both functional and nonfunctional genomic elements in the testis compared to the other organs. To elucidate the cellular source and mechanisms underlying this promiscuous transcription in the testis, we generated deep coverage RNA-Seq data for all major testis cell types as well as epigenetic data (DNA and histone methylation) using the mouse as model system. The integration of these complete dataset revealed that meiotic and especially post-meiotic germ cells are the major contributors to the widespread functional and nonfunctional transcriptome complexity of the testis, and that this "promiscuous" spermatogenic transcription is resulting, at least partially, from an overall transcriptionally permissive chromatin state. We hypothesize that this particular open state of the chromatin results from the extensive chromatin remodeling that occurs during spermatogenesis which ultimately leads to the replacement of histones by protamines in the mature spermatozoa. Our results have important functional and evolutionary implications (e.g., regarding new gene birth and testicular gene expression evolution).Generally, these three large-scale projects of my thesis provide complete and massive datasets that constitute valuables resources for further functional and evolutionary analyses of mammalian genomes.

ANIBAL, stable isotope-based quantitative proteomics by aniline and benzoic acid labeling of amino and carboxylic groups

Identification and relative quantification of hundreds to thousands of proteins within complex biological samples have become realistic with the emergence of stable isotope labeling in combination with high throughput mass spectrometry. However, all current chemical approaches target a single amino acid functionality (most often lysine or cysteine) despite the fact that addressing two or more amino acid side chains would drastically increase quantifiable information as shown by in silico analysis in this study. Although the combination of existing approaches, e.g. ICAT with isotope-coded protein labeling, is analytically feasible, it implies high costs, and the combined application of two different chemistries (kits) may not be straightforward. Therefore, we describe here the development and validation of a new stable isotope-based quantitative proteomics approach, termed aniline benzoic acid labeling (ANIBAL), using a twin chemistry approach targeting two frequent amino acid functionalities, the carboxylic and amino groups. Two simple and inexpensive reagents, aniline and benzoic acid, in their (12)C and (13)C form with convenient mass peak spacing (6 Da) and without chromatographic discrimination or modification in fragmentation behavior, are used to modify carboxylic and amino groups at the protein level, resulting in an identical peptide bond-linked benzoyl modification for both reactions. The ANIBAL chemistry is simple and straightforward and is the first method that uses a (13)C-reagent for a general stable isotope labeling approach of carboxylic groups. In silico as well as in vitro analyses clearly revealed the increase in available quantifiable information using such a twin approach. ANIBAL was validated by means of model peptides and proteins with regard to the quality of the chemistry as well as the ionization behavior of the derivatized peptides. A milk fraction was used for dynamic range assessment of protein quantification, and a bacterial lysate was used for the evaluation of relative protein quantification in a complex sample in two different biological states

Functional interaction between the estrogen receptor and CTF1: analysis of the vitellogenin gene B1 promoter in yeast.

Eukaryotic gene expression depends on a complex interplay between the transcriptional apparatus and chromatin structure. We report here a yeast model system for investigating the functional interaction between the human estrogen receptor (hER) and CTF1, a member of the CTF/NFI transcription factor family. We show that a CTF1-fusion protein and the hER transactivate a synthetic promoter in yeast in a synergistic manner. This interaction requires the proline-rich transactivation domain of CTF1. When the natural estrogen-dependent vitellogenin B1 promoter is tested in yeast, CTF1 and CTF1-fusion proteins are unable to activate transcription, and no synergy is observed between hER, which activates the B1 promoter, and these factors. Chromatin structure analysis on this promoter reveals positioned nucleosomes at -430 to -270 (+/-20 bp) and at -270 to - 100 (+/-20 bp) relative to the start site of transcription. The positions of the nucleosomes remain unchanged upon hormone-dependent transcriptional activation of the promoter, and the more proximal nucleosome appears to mask the CTF/NFI site located at - 101 to -114. We conclude that a functional interaction of hER with the estrogen response element located upstream of a basal promoter occurs in yeast despite the nucleosomal organization of this promoter, whereas the interaction of CTF1 with its target site is apparently precluded by a nucleosome.

Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.

The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.

Species selectivity of mixed-lineage leukemia/trithorax and HCF proteolytic maturation pathways.

Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1(PRO) repeats. We demonstrate here that the Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.

Alkylpurine-DNA-N-glycosylase confers resistance to temozolomide in xenograft models of glioblastoma multiforme and is associated with poor survival in patients.

Glioblastoma multiforme (GBM) is the most common and lethal of all gliomas. The current standard of care includes surgery followed by concomitant radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). O⁶-methylguanine-DNA methyltransferase (MGMT) repairs the most cytotoxic of lesions generated by TMZ, O⁶-methylguanine. Methylation of the MGMT promoter in GBM correlates with increased therapeutic sensitivity to alkylating agent therapy. However, several aspects of TMZ sensitivity are not explained by MGMT promoter methylation. Here, we investigated our hypothesis that the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG), which repairs the cytotoxic lesions N³-methyladenine and N⁷-methylguanine, may contribute to TMZ resistance. Silencing of APNG in established and primary TMZ-resistant GBM cell lines endogenously expressing MGMT and APNG attenuated repair of TMZ-induced DNA damage and enhanced apoptosis. Reintroducing expression of APNG in TMZ-sensitive GBM lines conferred resistance to TMZ in vitro and in orthotopic xenograft mouse models. In addition, resistance was enhanced with coexpression of MGMT. Evaluation of APNG protein levels in several clinical datasets demonstrated that in patients, high nuclear APNG expression correlated with poorer overall survival compared with patients lacking APNG expression. Loss of APNG expression in a subset of patients was also associated with increased APNG promoter methylation. Collectively, our data demonstrate that APNG contributes to TMZ resistance in GBM and may be useful in the diagnosis and treatment of the disease.