Transcriptional regulation of the mannosyltransferase-encoding gene pigm in inherited glycosylphosphatidylinositol (GPI) deficiency

RESUMO:O glicosilfosfatidilinositol (GPI) �� um complexo glicolip��dico utlizado por dezenas de prote��nas, o qual medeia a sua ancoragem �� superf��cie da c��lula. Prote��nas de superf��cie celular ancoradas a GPI apresentam v��rias fun����es essenciais para a manuten����o celular. A defici��ncia na s��ntese de GPI �� o que caracteriza principalmente a defici��ncia heredit��ria em GPI, um grupo de doen��as autoss��micas raras que resultam de muta����es nos genes PIGA, PIGL, PIGM, PIGV, PIGN, PIGO e PIGT, os quais sao indispens��veis para a bioss��ntese do GPI. Uma muta����o pontual no motivo rico em GC -270 no promotor de PIGM impede a liga����o do factor de transcri����o (FT) Sp1 �� sua sequ��ncia de reconhecimento, impondo a compacta����o da cromatina, associada �� hipoacetila����o de histonas, e consequentemente, impedindo a transcri����o de PIGM. Desta forma, a adi����o da primeira manose ao GPI �� comprometida, a s��ntese de GPI diminui assim como as prote��nas ligadas a GPI �� superficie das c��lulas. Pacientes com Defici��ncia Heredit��ria em GPI-associada a PIGM apresentam trombose e epilesia, e aus��ncia de hem��lise intravascular e anemia, sendo que estas duas ��ltimas caracter��sticas definem a Hemoglobin��ria Parox��stica Nocturna (HPN), uma doen��a rara causada por muta����es no gene PIGA. Embora a muta����o que causa IGD seja constitutiva e esteja presente em todos os tecidos, o grau de defici��ncia em GPI varia entre c��lulas do mesmo tecido e entre c��lulas de tecidos diferentes. Por exemplo nos granul��citos e linf��citos B a defici��ncia em GPI �� muito acentuada mas nos linf��citos T, fibroblastos, plaquetas e eritr��citos �� aproximadamente normal, da�� a aus��ncia de hem��lise intravascular. Os eventos transcricionais que est��o na base da express��o diferencial da ��ncora GPI nas c��lulas hematopoi��ticas s��o desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os n��veis de PIGM mRNA variam entre c��lulas prim��rias hematopoi��ticas normais. Adicionalmente, a configura����o dos nucleossomas no promotor de PIGM �� mais compacta em c��lulas B do que em c��lulas eritr��ides e tal est�� correlacionado com os n��veis de express��o de PIGM, isto ��, inferior nas c��lulas B. A presen��a de v��rios motivos de liga����o para o FT espec��fico da linhagem megacarioc��tica-eritr��ide GATA-1 no promotor de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcri����o. Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel repressor em vez de activador da express��o de PIGM. Resultados preliminares sugerem que KLF1, um factor de transcri����o restritamente eritr��ide, regula a transcri����o de PIGM independentemente do motivo -270GC. Em segundo lugar, a investiga����o do papel dos FTs Sp demonstrou que Sp1 medeia directamente a transcri����o de PIGM em ambas as c��lulas B e eritr��ide. Curiosamente, ao contr��rio do que acontece nas c��lulas B, em que a transcri����o de PIGM requer a liga����o do FT geral Sp1 ao motivo -270GC, nas c��lulas eritr��ides Sp1 regula a transcri����o de PIGM ao ligar-se a montante e n��o ao motivo -270GC. Para al��m disso, demonstrou-se que Sp2 n��o �� um regulador directo da transcri����o de PIGM quer nas c��lulas B quer nas c��lulas eritr��ides. Estes resultados explicam a aus��ncia de hem��lise intravascular nos doentes com IGD associada a PIGM, uma das principais caracter��sticas que define a HPN. Por ��ltimo, resultados preliminares mostraram que a repress��o da transcri����o de PIGM devida �� muta����o patog��nica -270C>G est�� associada com a diminui����o da frequ��ncia de interac����es gen��micas em cis entre PIGM e os seus genes ���vizinhos���, sugerindo adicionalmente que a regula����o de PIGM e desses genes �� partilhada. No seu conjunto, os resultados apresentados nesta tese contribuem para o conhecimento do controlo transcricional de um gene housekeeping, espec��fico-detecido, por meio de FTs gen��ricos e espec��ficos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of proteins for cell surface anchoring. GPI-anchored proteins have various functions that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis. A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction associated with histone hypoacetylation, thus abrogating transcription of PIGM. As a consequence of PIGM transcriptional repression, addition of the first mannose residue onto the GPI core and thus GPI production are impaired; and expression of GPI-anchored proteins on the surface of cells is severely impaired. Patients with PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA. Although the disease-causing mutation in IGD is constitutional and present in all tissues, the degree of GPI deficiency is variable and differs between cells of the same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the haematopoietic cells of PIG-M-associated IGD are not known and constitute the general aim of this thesis. Firstly, I found that PIGM mRNA levels are variable amongst normal primary haematopoietic cells. In addition, the nucleosome configuration in the promoter of PIGM is more compacted in B cells than in erythroid cells and this correlated with the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a repressor rather than an activator of PIGM expression. Preliminary data suggested that KLF1, an erythroid-specific TF, regulates PIGM transcription but independently of the -270GC motif. Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B cells in which active PIGM transcription requires binding of the generic TF Sp1 to the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by binding upstream of but not to the -270GC-rich motif. Additionally, I showed that Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells. These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a shared regulatory link between these genes and PIGM. Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and generic TFs.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

Evolutionary switch and genetic convergence on rbcL following the evolution of C4 photosynthesis.

Rubisco is responsible for the fixation of CO2 into organic compounds through photosynthesis and thus has a great agronomic importance. It is well established that this enzyme suffers from a slow catalysis, and its low specificity results into photorespiration, which is considered as an energy waste for the plant. However, natural variations exist, and some Rubisco lineages, such as in C4 plants, exhibit higher catalytic efficiencies coupled to lower specificities. These C4 kinetics could have evolved as an adaptation to the higher CO2 concentration present in C4 photosynthetic cells. In this study, using phylogenetic analyses on a large data set of C3 and C4 monocots, we showed that the rbcL gene, which encodes the large subunit of Rubisco, evolved under positive selection in independent C4 lineages. This confirms that selective pressures on Rubisco have been switched in C4 plants by the high CO2 environment prevailing in their photosynthetic cells. Eight rbcL codons evolving under positive selection in C4 clades were involved in parallel changes among the 23 independent monocot C4 lineages included in this study. These amino acids are potentially responsible for the C4 kinetics, and their identification opens new roads for human-directed Rubisco engineering. The introgression of C4-like high-efficiency Rubisco would strongly enhance C3 crop yields in the future CO2-enriched atmosphere.

Expression analysis of the AtPHO1 gene family in Arabidopsis thaliana and the involvement of AtPHO1 in the regulation of stomatal movements

R��sum�� Le transfert du phosphate des racines vers les feuilles s'effectue par la voie du xyl��me. Il a ��t�� pr��c��demment d��montr�� que la prot��ine AtPHO1 ��tait indispensable au transfert du phosphate dans les vaisseaux du xyl��me des racines chez la plante mod��le Arabidopsis thaliana. Le s��quen��age et l'annotation du g��nome d'Arabidopsis ont permis d'identifier dix s��quences pr��sentant un niveau de similarit�� significatif avec le g��ne AtPHO1 et constituant une nouvelle famille de g��ne appel�� la famille de AtPHO1. Bas��e sur une ��tude mol��culaire et g��n��tique, cette th��se apporte des ��l��ments de r��ponse pour d��terminer le r��le des membres de ia famille de AtPHO1 chez Arabidopsis, inconnue �� ce jour. Dans un premier temps, une analyse bioinformatique des s��quences prot��iques des membres de la famille de AtPHO1 a r��v��l�� la pr��sence dans leur r��gion N-terminale d'un domaine nomm�� SPX. Ce dernier est conserv�� parmi de nombreuses prot��ines impliqu��es dans l'hom��ostasie du phosphate chez la levure, renfor��ant ainsi l'hypoth��se que les membres de la famille de AtPHO1 auraient comme AtPHO1 un r��le dans l'��quilibre du phosphate dans la plante. En parall��le, la localisation tissulaire de l'expression des g��nes AtPHO dans Arabidopsis a ��t�� identifi��e par l'analyse de plantes transg��niques exprimant le g��ne rapporteur uidA sous le contr��le des promoteurs respectifs des g��nes AtPHO. Un profil d'expression de chaque g��ne AtPHO au cours du d��veloppement de la plante a ��t�� obtenu. Une expression pr��dominante au niveau des tissus vasculaires des racines, des feuilles, des tiges et des fleurs a ��t�� observ��e, sugg��rant que les g��nes AtPHO pourraient avoir des fonctions redondantes au niveau du transfert de phosphate dans le cylindre vasculaire de ces diff��rents organes. Toutefois, plusieurs r��gions promotrices des g��nes AtPHO contr��lent ��galement un profil d'expression GUS non-vasculaire, indiquant un r��le putatif des g��nes AtPHO dans l'acquisition ou le recyclage de phosphate dans la plante. Dans un deuxi��me temps, l'analyse de l'expression des g��nes AtPHO durant une carence en phosphate a ��tabli que seule l'expression des g��nes AtPHO1, AtPHO1; H1 et AtPHO1; H10 est r��gul��e par cette carence. Une ��tude approfondie de leur expression en r��ponse �� des traitements affectant l'hom��ostasie du phosphate dans la plante a ensuite d��montr�� leur r��gulation par diff��rentes voies de signalisation. Ensuite, une analyse d��taill��e de la r��gulation de l'expression du g��ne AtPHO1; H1O dans des feuilles d'Arabidopsis bless��es ou d��shydrat��es a r��v��l�� que ce g��ne constitue le prem��er g��ne marqueur d'une nouvelle voie de signalisation induite par l'OPDA, pas par le JA et d��pendante de la prot��ine COI1. Ces r��sultats d��montrent pour la premi��re fois que l'OPDA et le JA peuvent activer diff��rents g��nes via des voies de signalisation d��pendantes de COI1. Enfin, cette th��se r��v��le l'identification d'un nouveau r��le de la prot��ine AtPHO1 dans la r��gulation de l'action de l'ABA au cours des processus de fermeture stomatique et de germination des graines chez Arabidopsis. Bien que les fonctions exactes des prot��ines AtPHO restent �� ��tre d��termin��es, ce travail de th��se sugg��re leur implication dans la propagation de diff��rents signaux dans la plante via la modulation du potentiel membranaire et/ou l'affectation de la composition en ions des cellules comme le font de nombreux transporteurs ou r��gulateur du transport d'ions. Summary Phosphate is transferred from the roots to the shoot via the xylem. The requirement for AtPHO1 protein to transfer phosphate to the xylem vessels of the root has been previously demonstrated in Arabidopsis thaliana. The sequencing and the annotation of the Arabidopsis genome had allowed the identification of ten sequences that show a significant level of similarity with the AtPHO1 gene. These 10 genes, of unknown functions, constitute a new gene family called the AtPHO1 gene family. Based on a molecular and genetics study, this thesis reveals some information needed to understand the role of the AtPHO1 family members in the plant Arabidopsis. First, a bioinformatics study revealed that the AtPHO sequences contained, in the N-terminal hydrophilic region, a motif called SPX and conserved among multiple proteins involved in phosphate homeostasis in yeast. This finding reinforces the hypothesis that all AtPHO1 family members have, as AtPHO1, a role in phosphate homeostasis. In parallel, we identified the pattern of expression of AtPHO genes in Arabidopsis via analysis of transgenic plants expressing the uidA reporter gene under the control of respective AtPHO promoter regions. The results exhibit a predominant expression of AtPHO genes in vascular tissues of all organs of the plant, implying that these AtPHO genes could have redundant functions in the transfer of phosphate to the vascular cylinder of various organs. The GUS expression pattern for several AtPHO promoter regions was also detected in non-vascular tissue indicating a broad role of AtPHO genes in the acquisition or in the recycling of phosphate in the plant. In a second step, the analysis of the expression of AtPHO genes during phosphate starvation established that only the expression of the AtPHO1, AtPHO1; H1 and AtPHO1; H10 genes were regulated by Pi starvation. Interestingly, different signalling pathways appeared to regulate these three genes during various treatments affecting Pi homeostasis in the plant. The third chapter presents a detailed analysis of the signalling pathways regulating the expression of the AtPHO1; H10 gene in Arabidopsis leaves during wound and dehydrated stresses. Surprisingly, the expression of AtPHO1; H10 was found to be regulated by OPDA (the precursor of JA) but not by JA itself and via the COI1 protein (the central regulator of the JA signalling pathway). These results demonstrated for the first time that OPDA and JA could activate distinct genes via COI1-dependent pathways. Finally, this thesis presents the identification of a novel role of the AtPHO1 protein in the regulation of ABA action in Arabidopsis guard cells and during seed germination. Although the exact role and function of AtPHO1 still need to be determined, these last findings suggest that AtPHO1 and by extension other AtPHO proteins could mediate the propagation of various signals in the plant by modulating the membrane potential and/or by affecting cellular ion composition, as it is the case for many ion transporters or regulators of ion transport.

Dictyostelium discoideum as an expression host for the circumsporozoite protein of Plasmodium falciparum.

We have used the cellular slime mold, Dictyostelium discoideum (Dd), to express the Plasmodium falciparum circumsporozoite protein (CS), a potential component of a subunit vaccine against malaria. This was accomplished via an expression vector based on the discoidin I-encoding gene promoter, in which we linked a sequence coding for a Dd leader peptide to the almost complete CS coding region (pEDII-CS). CS production at both the mRNA and protein levels is induced by starving cells in a simple phosphate buffer. Variation in pH or cell density does not seem to influence CS synthesis. CS-producing cells can be grown either on their normal substrate, bacteria, or on a semi-synthetic media, without affecting CS accumulation level. The CS produced in Dd seems similar to the natural parasite protein as judged by its size and epitope recognition by a panel of monoclonal antibodies. We constructed a second expression vector in which the CS is under the control of a Dd ras promoter. CS accumulation can then be induced by external addition of cAMP. Such a tightly regulated promoter may allow expression of proteins potentially toxic to the cell. Thus, Dd could be a useful eukaryotic system to produce recombinant proteins, in particular from human or animal parasites like P. falciparum.

Role of microRNAs in the pathogenesis of Ewing's sarcoma

SummaryEwing's sarcoma family tumors (ESFT) are the second most frequent cancer of bone in adolescents and young adults. ESFT are characterized by a chromosomal translocation that involves the 5' segment of the EWSR1 gene and the 3' segment of an ets transcription factor family member gene. In 85% of cases the chromosomal translocation generates the fusion protein EWSR1-FLI-1. Recent work from our laboratory identified mesenchymal stem cells (MSC) as the putative cell of origin of ESFT and characterized a CD133+ subpopulation of ESFT cells with tumor initating and self-renewal capacity, known as cancer stem cells (CSC). MicroRNAs (miRNAs) are small non-coding RNA that regulate protein expression at the post-transcriptional level by either repressing translation or destabilizing mRNA. MiRNAs participate in several biological processes including cell proliferation and differentiation. We used miRNA expression profile comparison between MSC and ESFT cell lines and CD133+ ESFT cells and CD133" ESFT cells to investigate the role of miRNAs in ESFT pathogenesis. MiRNA expression profile comparison of MSC and ESFT cell lines identified 35 differentially expressed miRNAs. Among these was down-regulation of let-7a which results, in part, by the direct repression of let-7a-l promoter by EWSR1-FLI-1. Overexpression of let-7a in ESFT cells blocked ESFT tumorigenesis through an High-motility group AT-hook2 (HMGA2)-mediated mechanism.MiRNA profiling of CD133+ ESFT and CD 133" ESFT cells revealed a broad repression of miRNAs in CD133+ ESFT mediated by down-regulation of TARBP2, a central regulator of the miRNA maturation pathway. Down-regulation of TARBP2 in ESFT cell lines results in a miRNA expression profile reminescent of that observed in CD133+ ESFT and associated with increased tumorigenicity. Enhancement of TARBP2 activity using the antibiotic enoxacin or overexpression of miRNA-143 or miRNA-145, two targets of TARBP2, impaired ESFT CSC self-renewal and block ESFT tumorigenicity. Moreover in vivo administration of synthetic let- 7a, miRNA-143 or miRNA-145 blocks ESFT tumor growth.Thus, dysregulation of miRNA expression is a key feature in ESFT pathogenesis and restoration of their expressions might be used as a new therapeutic tool.R��sum��Le sarcome d'Ewing est la deuxi��me tumeur osseuse la plus fr��quente chez l'enfant et le jeune adolescent. Le sarcome d'Ewing est caract��ris�� par une translocation chromosomique qui produit une prot��ine de fusion EWSR1-FLI-1. Des r��cents travaux ont identifi�� les cellules m��senchymateuses souches (MSC) comme ��tant les cellules �� l'origine du sarcome d'Ewing ainsi qu'une sous-population de cellules exprimant le marqueur CD 133, dans le sarcome d'Ewing connu comme les cellules canc��reuses souches (CSC). Ces cellules ont la capacit�� d'initier la croissance tumorale et poss��dent des propri��t��s d'auto-renouvellement. Les microRNAs (miRNAs) sont de petits ARN qui ne codent pas pour des prot��ines et qui contr��lent l'expression des prot��ines en bloquant la traduction ou en d��gradant l'ARNm. Les miRNAs participent �� diff��rents processus biologiques comme la prolif��ration et la diff��renciation cellulaires.Le but de ce travail est d'��tudier le r��le des miRNAs dans le sarcome d'Ewing. Un profil d'expression de miRNAs entre les MSC et des lign��es cellulaires de sarcome d'Ewing a mis en ��vidence 35 miRNAs diff��remment exprim��s. Parmi ceux-ci, la r��pression de let-7a est li��e �� la r��pression directe du promoteur de let-7a-l par EWSR-FLI-1. La sur-expression de let-7a dans des lign��es cellulaires de sarcome d'Ewing inhibe leur croissance tumorale. Cette inhibition de croissance tumorale est r��gul��e par la prot��ine high-motility group AT-hook2 (HMGA2).Un profil d'expression de miRNAs entre les cellules du sarcome d'Ewing CD133+ et CD133" montre une sous-expression d'un grand nombre de miRNAs dans les cellules CD133+ par rapport aux cellules CD133". Cette diff��rence d'expression de miRNAs est due �� la r��pression du g��ne TARBP2 qui participe �� la maturation des miRNAs. La suppression de TARBP2 dans des cellules d'Ewing induit un profil d'expression de miRNAs similaire aux cellules CD133+ du sarcome d'Ewing et augmente la tumorigen��se des lign��es cellulaires. De plus l'utilisation d'enoxacin, une mol��cule qui augmente l'activit�� de TARBP2 ou la sur- expression des miRNA143 ou miRNA-145 dans les CSC du sarcome d'Ewing bloque l'auto- renouvellement des cellules et la croissance tumorale. Finalement, l'administration de let-7a, miRNA-143 ou miRNA-145, dans des souris bloque la croissance du sarcome d'Ewing. Ces r��sultats indiquent que la dysr��gulation des miRNAs participe �� la pathogen��se du sarcome d'Ewing et que les miRNAs peuvent ��tre utilis��s comme des agents th��rapeutiques.

Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.

Selection of patients with germline MLH1 mutated Lynch syndrome by determination of MLH1 methylation and BRAF mutation.

Lynch syndrome is one of the most common hereditary colorectal cancer (CRC) syndrome and is caused by germline mutations of MLH1, MSH2 and more rarely MSH6, PMS2, MLH3 genes. Whereas the absence of MSH2 protein is predictive of Lynch syndrome, it is not the case for the absence of MLH1 protein. The purpose of this study was to develop a sensitive and cost effective algorithm to select Lynch syndrome cases among patients with MLH1 immunohistochemical silencing. Eleven sporadic CRC and 16 Lynch syndrome cases with MLH1 protein abnormalities were selected. The BRAF c.1799T> A mutation (p.Val600Glu) was analyzed by direct sequencing after PCR amplification of exon 15. Methylation of MLH1 promoter was determined by Methylation-Sensitive Single-Strand Conformation Analysis. In patients with Lynch syndrome, there was no BRAF mutation and only one case showed MLH1 methylation (6%). In sporadic CRC, all cases were MLH1 methylated (100%) and 8 out of 11 cases carried the above BRAF mutation (73%) whereas only 3 cases were BRAF wild type (27%). We propose the following algorithm: (1) no further molecular analysis should be performed for CRC exhibiting MLH1 methylation and BRAF mutation, and these cases should be considered as sporadic CRC; (2) CRC with unmethylated MLH1 and negative for BRAF mutation should be considered as Lynch syndrome; and (3) only a small fraction of CRC with MLH1 promoter methylation but negative for BRAF mutation should be true Lynch syndrome patients. These potentially Lynch syndrome patients should be offered genetic counselling before searching for MLH1 gene mutations.

Mutation screening of the glutamate cysteine ligase modifier (GCLM) gene in patients with schizophrenia.

BACKGROUND: Experimental evidences show that glutathione and its rate-limiting synthesizing enzyme, the glutamate-cysteine ligase (GCL), are involved in the pathogenesis of schizophrenia. Furthermore, genetic association has been previously reported between two single nucleotide polymorphisms lying in noncoding regions of glutamate cysteine ligase modifier (GCLM) gene, which specifies for the modifier subunit of GCL and schizophrenia. OBJECTIVE: We wanted to investigate the presence of GCLM true functional mutations, likely in linkage disequilibrium with the previously identified single nucleotide polymorphism alleles, in the same set of cases that allowed the detection of the original association signal. METHODS: We screened all the coding regions of GCLM and their intronic flanking vicinities in 353 patients with schizophrenia by direct DNA sequencing. RESULTS: Ten sequence variations were identified, five of which were not previously described. None of these DNA changes was within the GCLM coding sequence and in-silico analysis failed to indicate functional impairment induced by these variations. Furthermore, screening of normal controls and downstream statistical analyses revealed no significant relationship of any of these DNA variants with schizophrenia. CONCLUSION: It is unlikely that functional mutations in the GCLM gene could play a major role in genetic predisposition to schizophrenia and further studies will be required to assess its etiological function in the disease.

EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

Gene expression mapping in neuropathic pain : molecular plasticity in nociceptors and superficial dorsal horn

RESUME : La douleur neuropathique est le r��sultat d'une l��sion ou d'un dysfonctionnement du syst��me nerveux. Les sympt��mes qui suivent la douleur neuropathique sont s��v��res et leur traitement inefficace. Une meilleure approche th��rapeutique peut ��tre propos��e en se basant sur les m��canismes pathologiques de la douleur neuropathique. Lors d'une l��sion p��riph��rique une douleur neuropathique peut se d��velopper et affecter le territoire des nerfs l��s��s mais aussi les territoires adjacents des nerfs non-l��s��s. Une hyperexcitabilit�� des neurones appara��t au niveau des ganglions spinaux (DRG) et de la corne dorsale (DH) de la moelle ��pini��re. Le but de ce travail consiste �� mettre en ��vidence les modifications mol��culaires associ��es aux nocicepteurs l��s��s et non-l��s��s au niveau des DRG et des laminae I et II de la corne dorsale, l�� o�� l'information nociceptive est int��gr��e. Pour ��tudier les changements mol��culaires li��s �� la douleur neuropathique nous utilisons le mod��le animal d'��pargne du nerf sural (spared nerve injury model, SNI) une semaine apr��s la l��sion. Pour la s��lection du tissu d'int��r��t nous avons employ�� la technique de la microdissection au laser, afin de s��lectionner une sous-population sp��cifique de cellules (notamment les nocicepteurs l��s��s ou non-l��s��s) mais ��galement de pr��lever le tissu correspondant dans les laminae superficielles. Ce travail est coupl�� �� l'analyse �� large spectre du transcriptome par puce ADN (microarray). Par ailleurs, nous avons ��tudi�� les courants ��lectriques et les propri��t��s biophysiques des canaux sodiques (Na,,ls) dans les neurones l��s��s et non-l��s��s des DRG. Aussi bien dans le syst��me nerveux p��riph��rique, entre les neurones l��s��s et non-l��s��s, qu'au niveau central avec les aires recevant les projections des nocicepteurs l��s��s ou non-l��s��s, l'analyse du transcriptome montre des diff��rences de profil d'expression. En effet, nous avons constat�� des changements transcriptionnels importants dans les nocicepteurs l��s��s (1561 g��nes, > 1.5x et pairwise comparaison > 77%) ainsi que dans les laminae correspondantes (618 g��nes), alors que ces modifications transcriptionelles sont mineures au niveau des nocicepteurs non-l��s��s (60 g��nes), mais important dans leurs laminae de projection (459 g��nes). Au niveau des nocicepteurs, en utilisant la classification par groupes fonctionnels (Gene Ontology), nous avons observ�� que plusieurs processus biologiques sont modifi��s. Ainsi des fonctions telles que la traduction des signaux cellulaires, l'organisation du cytosquelette ainsi que les m��canismes de r��ponse au stress sont affect��s. Par contre dans les neurones non-l��s��s seuls les processus biologiques li��s au m��tabolisme et au d��veloppement sont modifi��s. Au niveau de la corne dorsale de la moelle, nous avons observ�� des modifications importantes des processus immuno-inflammatoires dans l'aire affect��e par les nerfs l��s��s et des changements associ��s �� l'organisation et la transmission synaptique au niveau de l'aire des nerfs non-l��s��s. L'analyse approfondie des canaux sodiques a d��montr�� plusieurs changements d'expression, principalement dans les neurones l��s��s. Les analyses fonctionnelles n'indiquent aucune diff��rence entre les densit��s de courant t��trodotoxine-sensible (TTX-S) dans les neurones l��s��s et non-l��s��s m��me si les niveaux d'expression des ARNm des sous-unit��s TTX-S sont modifi��s dans les neurones l��s��s. L'inactivation basale d��pendante du voltage des canaux t��trodotoxine-insensible (TTX-R) est d��plac��e vers des potentiels positifs dans les cellules l��s��es et non-l��s��es. En revanche la vitesse de r��cup��ration des courants TTX-S et TTX-R apr��s inactivation est acc��l��r��e dans les neurones l��s��s. Ces changements pourraient ��tre �� l'origine de l'alt��ration de l'activit�� ��lectrique des neurones sensoriels dans le contexte des douleurs neuropathiques. En r��sum��, ces r��sultats sugg��rent l'existence de m��canismes diff��renci��s affectant les neurones l��s��s et les neurones adjacents non-l��s��s lors de la mise en place la douleur neuropathique. De plus, les changements centraux au niveau de la moelle ��pini��re qui surviennent apr��s l��sion sont probablement int��gr��s diff��remment selon la perception de signaux des neurones p��riph��riques l��s��s ou non-l��s��s. En conclusion, ces modulations complexes et distinctes sont probablement des acteurs essentiels impliqu��s dans la gen��se et la persistance des douleurs neuropathiques. ABSTRACT : Neuropathic pain (NP) results from damage or dysfunction of the peripheral or central nervous system. Symptoms associated with NP are severe and difficult to treat. Targeting NP mechanisms and their translation into symptoms may offer a better therapeutic approach.Hyperexcitability of the peripheral and central nervous system occurs in the dorsal root ganglia (DRG) and the dorsal horn (DH) of the spinal cord. We aimed to identify transcriptional variations in injured and in adjacent non-injured nociceptors as well as in corresponding laminae I and II of DH receiving their inputs.We investigated changes one week after the injury induced by the spared nerve injury model of NP. We employed the laser capture microdissection (LCM) for the procurement of specific cell-types (enrichment in nociceptors of injured/non-injured neurons) and laminae in combination with transcriptional analysis by microarray. In addition, we studied function��l properties and currents of sodium channels (Nav1s) in injured and neighboring non-injured DRG neurons.Microarray analysis at the periphery between injured and non-injured DRG neurons and centrally between the area of central projections from injured and non-injured neurons show significant and differential expression patterns. We reported changes in injured nociceptors (1561 genes, > 1.5 fold, >77% pairwise comparison) and in corresponding DH laminae (618 genes), while less modifications occurred in non-injured nociceptors (60 genes) and in corresponding DH laminae (459 genes). At the periphery, we observed by Gene Ontology the involvement of multiple biological processes in injured neurons such as signal transduction, cytoskeleton organization or stress responses. On contrast, functional overrepresentations in non-injured neurons were noted only in metabolic or developmentally related mechanisms. At the level of superficial laminae of the dorsal horn, we reported changes of immune and inflammatory processes in injured-related DH and changes associated with synaptic organization and transmission in DH corresponding to non-injured neurons. Further transcriptional analysis of Nav1s indicated several changes in injured neurons. Functional analyses of Nav1s have established no difference in tetrodotoxin-sensitive (TTX-S) current densities in both injured and non-injured neurons, despite changes in TTX-S Nav1s subunit mRNA levels. The tetrodotoxin-resistant (TTX-R) voltage dependence of steady state inactivation was shifted to more positive potentials in both injured and non-injured neurons, and the rate of recovery from inactivation of TTX-S and TTX-R currents was accelerated in injured neurons. These changes may lead to alterations in neuronal electrogenesis. Taken together, these findings suggest different mechanisms occurring in the injured neurons and the adjacent non-injured ones. Moreover, central changes after injury are probably driven in a different manner if they receive inputs from injured or non-injured neurons. Together, these distinct and complex modulations may contribute to NP.

Small and long non-coding RNAs in cardiac homeostasis and regeneration

Cardiovascular diseases and in particular heart failure are major causes of morbidity and mortality in the Western world. Recently, the notion of promoting cardiac regeneration as a means to replace lost cardiomyocytes in the damaged heart has engendered considerable research interest. These studies envisage the utilization of both endogenous and exogenous cellular populations, which undergo highly specialized cell fate transitions to promote cardiomyocyte replenishment. Such transitions are under the control of regenerative gene regulatory networks, which are enacted by the integrated execution of specific transcriptional programs. In this context, it is emerging that the non-coding portion of the genome is dynamically transcribed generating thousands of regulatory small and long non-coding RNAs, which are central orchestrators of these networks. In this review, we discuss more particularly the biological roles of two classes of regulatory non-coding RNAs, i.e. microRNAs and long non-coding RNAs, with a particular emphasis on their known and putative roles in cardiac homeostasis and regeneration. Indeed, manipulating non-coding RNA-mediated regulatory networks could provide keys to unlock the dormant potential of the mammalian heart to regenerate. This should ultimately improve the effectiveness of current regenerative strategies and discover new avenues for repair. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Molecular screening of ADAMTSL2 gene in 33 patients reveals the genetic heterogeneity of geleophysic dysplasia.

Background Geleophysic dysplasia (GD, OMIM 231050) is an autosomal recessive disorder characterised by short stature, small hands and feet, stiff joints, and thick skin. Patients often present with a progressive cardiac valvular disease which can lead to an early death. In a previous study including six GD families, we have mapped the disease gene on chromosome 9q34.2 and identified mutations in the A Disintegrin And Metalloproteinase with Thrombospondin repeats-like 2 gene (ADAMTSL2). Methods Following this study, we have collected the samples of 30 additional GD families, including 33 patients and identified ADAMTSL2 mutations in 14/33 patients, comprising 13 novel mutations. The absence of mutation in 19 patients prompted us to compare the two groups of GD patients, namely group 1, patients with ADAMTSL2 mutations (n=20, also including the 6 patients from our previous study), and group 2, patients without ADAMTSL2 mutations (n=19). Results The main discriminating features were facial dysmorphism and tip-toe walking, which were almost constantly observed in group 1. No differences were found concerning heart involvement, skin thickness, recurrent respiratory and ear infections, bronchopulmonary insufficiency, laryngo-tracheal stenosis, deafness, and radiographic features. Conclusions It is concluded that GD is a genetically heterogeneous condition. Ongoing studies will hopefully lead to the identification of another disease gene.

Glycogen synthase 2 is a novel target gene of peroxisome proliferator-activated receptors.

Glycogen synthase 2 (Gys-2) is the ratelimiting enzyme in the storage of glycogen in liver and adipose tissue, yet little is known about regulation of Gys-2 transcription. The peroxisome proliferator-activated receptors (PPARs) are transcription factors involved in the regulation of lipid and glucose metabolism and might be hypothesized to govern glycogen synthesis as well. Here, we show that Gys-2 is a direct target gene of PPARalpha, PPARbeta/delta and PPARgamma. Expression of Gys-2 is significantly reduced in adipose tissue of PPARalpha-/-, PPARbeta/delta-/- and PPARgamma+/- mice. Furthermore, synthetic PPARbeta/delta, and gamma agonists markedly up-regulate Gys-2 mRNA and protein expression in mouse 3T3-L1 adipocytes. In liver, PPARalpha deletion leads to decreased glycogen levels in the refed state, which is paralleled by decreased expression of Gys-2 in fasted and refed state. Two putative PPAR response elements (PPREs) were identified in the mouse Gys-2 gene: one in the upstream promoter (DR-1prom) and one in intron 1 (DR-1int). It is shown that DR-1int is the response element for PPARs, while DR-1prom is the response element for Hepatic Nuclear Factor 4 alpha (HNF4alpha). In adipose tissue, which does not express HNF4alpha, DR-1prom is occupied by PPARbeta/delta and PPARgamma, yet binding does not translate into transcriptional activation of Gys-2. Overall, we conclude that mouse Gys-2 is a novel PPAR target gene and that transactivation by PPARs and HNF4alpha is mediated by two distinct response elements.

Structural organization of alpha-subunit from purified and microsomal toad kidney (Na+ + K+)-ATPase as assessed by controlled trypsinolysis

The membrane organization of the alpha-subunit of purified (Na+ + K+)-ATPase ((Na+ + K+)-dependent adenosine triphosphate phosphorylase, EC 3.6.1.3) and of the microsomal enzyme of the kidney of the toad Bufo marinus was compared by using controlled trypsinolysis. With both enzyme preparations, digestions performed in the presence of Na+ yielded a 73 kDa fragment and in the presence of K+ a 56 kDa, a 40 kDa and small amounts of a 83 kDa fragment from the 96 kDa alpha-subunit. In contrast to mammalian preparations (J��rgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415), trypsinolysis of the purified amphibian enzyme led to a biphasic loss of (Na+ + K+)-ATPase activity in the presence of both Na+ and K+. These data could be correlated with an early rapid cleavage of 3 kDa from the alpha-subunit in both ionic conditions and a slower degradation of the remaining 93 kDa polypeptide. On the other hand, in the microsomal enzyme, a 3 kDa shift of the alpha-subunit could only be produced in the presence of Na+. Our data indicate that (1) purification of the amphibian enzyme with detergent does not influence the overall topology of the alpha-subunit but produces a distinct structural alteration of its N-terminus and (2) the amphibian kidney enzyme responds to cations with similar conformational transitions as the mammalian kidney enzyme. In addition, anti alpha-serum used on digested enzyme samples revealed on immunoblots that the 40 kDa fragment was better recognized than the 56 kDa fragment. It is concluded that the NH2-terminal of the alpha-subunit contains more antigenic sites than the COOH-terminal domain in agreement with the results of Farley et al. (Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906).