Several lines of schizophrenia (SZ) research suggest that a functional downregulation of the prefrontal cortex GABAergic neuronal system is mediated by a promoter hypermethylation, presumably catalyzed by an increase in DNA-methyltransferase-1 (DNMT-1) expression. This promoter hypermethylation may be mediated not only by DNMT-1 but also by an entire family of de novo DNA-methyltransferases, such as DNA-methyltransferase-3a (DNMT-3a) and -3b (DNMT-3b). To verify the existence of an overexpression of DNMT-3a and DNMT-3b in the brain of schizophrenia patients (SZP), we compared their mRNA expression in Brodmann's area 10 (BA10) and in the caudate nucleus and putamen obtained from the Harvard Brain Tissue Resource Center (Belmont, MA) from both nonpsychiatric subjects (NPS) and SZP. Our results demonstrate that DNMT-3a and DNMT-1 are expressed and co-localize in distinct GABAergic neuron populations whereas DNMT-3b mRNA is virtually undetectable. We also found that unlike DNMT-1, which is frequently overexpressed in telencephalic GABAergic neurons of SZP, DNMT-3a mRNA is overexpressed only in layer I and II GABAergic interneurons of BA10. To ascertain whether these DNMT expression differences observed in brain tissue could also be detected in peripheral tissues, we studied whether DNMT-1 and DNMT-3a mRNAs were overexpressed in peripheral blood lymphocytes (PBL) of SZP. Both DNMT-1 and DNMT-3a mRNAs are expressed in the PBL and although DNMT-3a mRNA levels in the PBL are approximately 1/10 of those of DNMT-1, the comparison of the PBL content in NPS and SZP showed a highly significant 2-fold increase of both DNMT-1 and DNMT-3a mRNA in SZP. These changes were unaffected by the dose, the duration, or the type of antipsychotic treatment. The upregulation of DNMT-1 and to a lesser extent that of DNMT-3a mRNA in PBL of SZP supports the concept that this readily available peripheral cell type can express an epigenetic variation of specific biomarkers relevant to SZ morbidity. Hence, PBL studies may become useful to investigate a diagnostic epigenetic marker of SZ morbidity.

O meduloblastoma (MB) é um câncer do sistema nervoso central, de origem embrionária, que surge no cerebelo. É o tumor maligno cerebral mais frequente na infância e corresponde a aproximadamente 20% de todos os tumores intracranianos pediátricos. Atualmente, o tratamento é realizado com cirurgia, quimioterapia e radioterapia e está relacionado com diversos efeitos colaterais em médio e longo prazo. Diversos fatores contribuem para o seu desenvolvimento e progressão, entre estes, alterações nas vias de sinalização, como a Sonic Hedgehog (SHH) e Wingless. As modificações nos padrões epigenéticos, como a metilação do DNA, tem também um papel central na biologia deste tumor. Tais alterações comprometem funções básicas da célula como o controle da proliferação, sobrevivência celular e apoptose. Drogas epigenéticas como os inibidores de DNA metiltransferases (DNMTs) têm demonstrado efeitos antineoplásicos e resultados promissores para terapia do câncer. A Zebularina é um inibidor de DNMTs, que consequentemente reduz a metilação do DNA, e tem se mostrado uma importante droga antitumoral, com baixa toxicidade e atividade adjuvante à quimioterapia em tumores quimio-resistentes. Diversos estudos têm descrito seus efeitos em diferentes tipos de neoplasias, entretanto, não há relatos da sua ação em MB. Sendo assim, o presente trabalho teve como objetivo analisar os potenciais efeitos antineoplásicos da Zebularina em quatro linhagens de MB pediátrico (DAOY, ONS-76, UW402 e UW473). Foi observado que o tratamento com a Zebularina promoveu inibição da proliferação celular e da capacidade clonogênica, aumentou o número de células apoptóticas e células na fase S do ciclo celular (p<0,05). Adicionalmente, o tratamento induziu um aumento na expressão proteica de p53, p21 e Bax e uma diminuição da ciclina A, Bcl-2 e Survivina. Além disso, quando combinada com o quimioterápico vincristina agiu de modo sinérgico; e de modo antagônico quando combinada com a cisplatina. Através de análises de expressão gênica em larga escala (plataforma Agilent de microarray), foi encontrada diferentes vias moduladas pela droga, incluindo a dos Receptores Toll-Like e o aumento dos genes SUFU e BATF2. Aqui, foi encontrado que a Zebularina pode modular a ativação da via SHH, reduzindo os níveis de SMO, de GLI1 e de um de seus alvos, o PTCH1; contudo sem alterar os níveis de SUFU. Confirmou-se que o gene BATF2 é induzido pela Zebularina e possui regiões ricamente metiladas. Além disso, a baixa expressão do gene BATF2 está associada à um pior prognóstico em MB. Todos esses dados sugerem que a Zebularina pode ser uma droga em potencial para o tratamento adjuvante do MB / Medulloblastoma (MB) is an embryonal cerebellum tumor. It is the most common brain malignancy in children and accounts for approximately 20% of all pediatric intracranial tumors. Currently, treatment consists of surgery, chemotherapy and radiation and is associated to medium- and long-term side effects. Several factors contribute to the development and progression of MB, for instance, alterations in signaling pathways, such as Sonic Hedgehog (SHH) and Wingless. Epigenetic changes in DNA methylation patterns also play a central role in the biology of this tumor. Such changes are able to alter basic cell functions, controlling cell proliferation, survival and apoptosis. Epigenetic drugs as DNA methyltransferases (DNMTs) inhibitors have shown anticancer effects and promising results for cancer therapy. Zebularine is a low toxicity DNMTs inhibitor that induces DNA demethylation and has been reported as an important antitumor drug with adjuvant activity to chemotherapy in chemoresistant tumors. Studies have described its effects on different types of cancer, however, there are not data concerning its action in MB. Therefore, this study aimed to analyze the potential anticancer effects of Zebularine in four pediatric MB lines (UW402, UW473, ONS- 76 and DAOY). It was observed that treatment with Zebularine promoted inhibition of cell proliferation and clonogenic capacity, increased the number of apoptosis rate and cells in S phase of the cycle (p <0.05). In addition, the treatment induced an increasing in the protein expression of p53, p21 and Bax and a decreasing in cyclin A, Survivin and Bcl-2. Also, when combined with the chemotherapeutic agent vincristine acted synergistically but resulted in antagonism when combined with cisplatin. Through large-scale gene expression analysis (Agilent microarray platform), it was found different pathways modulated by Zebularine, including the Toll-Like Receptors pathway and the overexpression of SUFU and BATF2 genes. Zebularine was able to modulate SHH pathway activation, by reducing levels of SMO, GLI1 and one of its targets, PTCH1, whereas there were no changes in SUFU levels. It was confirmed that the gene BATF2 is induced by Zebularine and contains regions richly methylated. In addition, BATF2 low expression is associated with a worse prognosis in MB. All these data suggest that Zebularine may be a potential drug for the adjuvant treatment of MB

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 69-72).

O meduloblastoma (MB) é um câncer do sistema nervoso central, de origem embrionária, que surge no cerebelo. É o tumor maligno cerebral mais frequente na infância e corresponde a aproximadamente 20% de todos os tumores intracranianos pediátricos. Atualmente, o tratamento é realizado com cirurgia, quimioterapia e radioterapia e está relacionado com diversos efeitos colaterais em médio e longo prazo. Diversos fatores contribuem para o seu desenvolvimento e progressão, entre estes, alterações nas vias de sinalização, como a Sonic Hedgehog (SHH) e Wingless. As modificações nos padrões epigenéticos, como a metilação do DNA, tem também um papel central na biologia deste tumor. Tais alterações comprometem funções básicas da célula como o controle da proliferação, sobrevivência celular e apoptose. Drogas epigenéticas como os inibidores de DNA metiltransferases (DNMTs) têm demonstrado efeitos antineoplásicos e resultados promissores para terapia do câncer. A Zebularina é um inibidor de DNMTs, que consequentemente reduz a metilação do DNA, e tem se mostrado uma importante droga antitumoral, com baixa toxicidade e atividade adjuvante à quimioterapia em tumores quimio-resistentes. Diversos estudos têm descrito seus efeitos em diferentes tipos de neoplasias, entretanto, não há relatos da sua ação em MB. Sendo assim, o presente trabalho teve como objetivo analisar os potenciais efeitos antineoplásicos da Zebularina em quatro linhagens de MB pediátrico (DAOY, ONS-76, UW402 e UW473). Foi observado que o tratamento com a Zebularina promoveu inibição da proliferação celular e da capacidade clonogênica, aumentou o número de células apoptóticas e células na fase S do ciclo celular (p<0,05). Adicionalmente, o tratamento induziu um aumento na expressão proteica de p53, p21 e Bax e uma diminuição da ciclina A, Bcl-2 e Survivina. Além disso, quando combinada com o quimioterápico vincristina agiu de modo sinérgico; e de modo antagônico quando combinada com a cisplatina. Através de análises de expressão gênica em larga escala (plataforma Agilent de microarray), foi encontrada diferentes vias moduladas pela droga, incluindo a dos Receptores Toll-Like e o aumento dos genes SUFU e BATF2. Aqui, foi encontrado que a Zebularina pode modular a ativação da via SHH, reduzindo os níveis de SMO, de GLI1 e de um de seus alvos, o PTCH1; contudo sem alterar os níveis de SUFU. Confirmou-se que o gene BATF2 é induzido pela Zebularina e possui regiões ricamente metiladas. Além disso, a baixa expressão do gene BATF2 está associada à um pior prognóstico em MB. Todos esses dados sugerem que a Zebularina pode ser uma droga em potencial para o tratamento adjuvante do MB / Medulloblastoma (MB) is an embryonal cerebellum tumor. It is the most common brain malignancy in children and accounts for approximately 20% of all pediatric intracranial tumors. Currently, treatment consists of surgery, chemotherapy and radiation and is associated to medium- and long-term side effects. Several factors contribute to the development and progression of MB, for instance, alterations in signaling pathways, such as Sonic Hedgehog (SHH) and Wingless. Epigenetic changes in DNA methylation patterns also play a central role in the biology of this tumor. Such changes are able to alter basic cell functions, controlling cell proliferation, survival and apoptosis. Epigenetic drugs as DNA methyltransferases (DNMTs) inhibitors have shown anticancer effects and promising results for cancer therapy. Zebularine is a low toxicity DNMTs inhibitor that induces DNA demethylation and has been reported as an important antitumor drug with adjuvant activity to chemotherapy in chemoresistant tumors. Studies have described its effects on different types of cancer, however, there are not data concerning its action in MB. Therefore, this study aimed to analyze the potential anticancer effects of Zebularine in four pediatric MB lines (UW402, UW473, ONS- 76 and DAOY). It was observed that treatment with Zebularine promoted inhibition of cell proliferation and clonogenic capacity, increased the number of apoptosis rate and cells in S phase of the cycle (p <0.05). In addition, the treatment induced an increasing in the protein expression of p53, p21 and Bax and a decreasing in cyclin A, Survivin and Bcl-2. Also, when combined with the chemotherapeutic agent vincristine acted synergistically but resulted in antagonism when combined with cisplatin. Through large-scale gene expression analysis (Agilent microarray platform), it was found different pathways modulated by Zebularine, including the Toll-Like Receptors pathway and the overexpression of SUFU and BATF2 genes. Zebularine was able to modulate SHH pathway activation, by reducing levels of SMO, GLI1 and one of its targets, PTCH1, whereas there were no changes in SUFU levels. It was confirmed that the gene BATF2 is induced by Zebularine and contains regions richly methylated. In addition, BATF2 low expression is associated with a worse prognosis in MB. All these data suggest that Zebularine may be a potential drug for the adjuvant treatment of MB

No description available.

No description available.

DNA methylation is a crucial, abundant mechanism of gene regulation in vertebrates. It is less prevalent in many other metazoan organisms and completely absent in some key model species, such as D. melanogaster and C. elegans. In this thesis we report on a comprehensive study of the pres- ence and absence of DNA methyltransferases (DNMTs) in 138 Ecdysozoa covering Arthropoda, Nematoda, Priapulida, Onychophora, and Tardigrada. We observe that loss of individual DNMTs independently occured multiple times across ecdysozoan phyla. In several cases, this resulted in a loss of DNA methylation. In vertebrates, however, there is no single species known which lost DNA methylation. Actually, DNA methylation was greatly expanded after the 1R/2R whole genome duplication (WGD) and became a genome-wide phe- nomena. In our study of vertebrates we are not looking for losses of DNA methyltransferases and DNA methylation but are rather interested in the gain of additional DNA methyltransferase genes. In vertebrates there were a number of WGD. Most vertebrates only underwent two WGD but in the teleost lineage a third round of WGD occured and in some groups, e.g. Salmoniformes and some Cypriniformes even a forth WGD occured. The Carp-specific WGD (4R) is one of the most recent vertebrate WGD and is estimated to have occured 12.4 mya. We performed the most comprehen- sive analysis of the evolution of DNA methyltransferases after vertebrate whole-genome duplications (WGD) so far. We were able to show that the conservation of duplicated DNMT3 genes in Salmoniformes is more diverse than previously believed. We were also able to identify DNA methyltrans- ferases in Cypriniformes which have, due to their recent WGD, quite com- plex genomes. Our results show that the patterns of retained and lost DNA methyltransferases after a forth round of WGD differ between Cypriniformes and Salmoniformes. We also proposed a new nomenclature for teleost DNMT genes which correctly represents the orthology of DNMT genes for all teleost species. Next to these purely computational projects we collaborated with the Aluru lab to investigate the effects of different disturbances on zebrafish DNA methylation. One disturbance is the inactivation of DNMT3aa and DNMT3ab as single knockouts as well as a double knockout. This was the first double knockout of DNMT genes in zebrafish which was ever generated. It allows us to study the subfunctionalization of the two DNMT3a genes their effect on genome-wide DNA methylation. Given our results we hypothesize that DNMT3aa and DNMT3ab can compensate for each other to a high de- gree. DNMT3a genes have likely been subfuntionalized but their loss can be compensated by DNMT3b genes. This compensation by DNMT3b genes works well enough that no notable phenotype can be observed in double knockout zebrafish but a difference is notable on the epigenome level. The second disturbance we studied is the exposure of zebrafish to the toxic chemi- cal PCB126. We detected a moderate level of DNA methylation changes and a much larger effect on gene expression. Similar to previous reports we find little correlation between DNA methylation and gene expression changes. Therefore, while PCB126 exposure has a negative effect on DNA methyla- tion it is likely that other gene regulatory mechanisms play a role as well, possibly even a greater one. How do genes evolve and how are genes regulated are two of the main questions of modern molecular biology. In this thesis we have tried to shed more light on both questions. we have broadly expanded the phylogenetic range of species with a manually curated set of DNA methyltransferases. We have done this for ecdysozoan species which have lost all DNA methylating enzymes as well as for teleost fish which acquired more than ten copies of the, originally, two genes. We were also able to generate new insight into the subfunctionalization of the DNA methylation machinery in zebrafish and how it reacts to environmental effects.:1 Introduction 1.1 Biological introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.2 Detecting DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . 7 2 Evolution of DNA methylation across Ecdysozoa 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3 Evolution of DNA methyltransferases after vertebrate whole genome duplications 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4 The effect of DNMT3aa and DNMT3ab knockout on DNA methyla- tion in zebrafish 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 4.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 5 Role of DNA methylation in altered testis gene expression patterns in adult zebrafish exposed to Pentachlorobiphenyl 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 5.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 6 Conclusions 6.1 Evolution of DNA methylation across Ecdysozoa . . . . . . . . . . . . . 95 6.2 Evolution of DNA methyltransferases after vertebrate whole genome duplications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 6.3 Role of DNA methylation in altered testis gene expression patterns in adult zebrafish (Danio rerio) exposed to Pentachlorobiphenyl (PCB 126). . . 107 6.4 Knockout of DNMT3aa and DNMT3ab in zebrafish (Danio rerio) . . . . . . 108 Bibliography 119

In multicellular organisms, all the cells are genetically identical but turn genes on or off at the right time to promote differentiation into specific cell types. The regulation of higher-order chromatin structure is essential for genome-wide reprogramming and for tissue-specific patterns of gene expression. The complexity of the genome is regulated by epigenetic mechanisms, which act at the level of DNA, histones, and nucleosomes. Epigenetic machinery is involved in many biological processes, including genomic imprinting, X-chromosome inactivation, heterochromatin formation, and transcriptional regulation, as well as DNA damage repair. In this review, we summarize the recent understanding of DNA methylation, cytosine derivatives, active and passive demethylation pathways as well as histone variants. DNA methylation is one of the well-characterized epigenetic signaling tools. Cytosine methylation of promoter regions usually represses transcription but methylation in the gene body may have a positive correlation with gene expression. The attachment of a methyl group to cytosine residue in the DNA sequence is catalyzed by enzymes of the DNA methyltransferase family. Recent studies have shown that the Ten-Eleven translocation family enzymes are involved in stepwise oxidation of 5-methylcytosine, creating new cytosine derivatives including 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Additionally, histone variants into nucleosomes create another strategy to regulate the structure and function of chromatin. The replacement of canonical histones with specialized histone variants regulates accessibility of DNA, and thus may affect multiple biological processes, such as replication, transcription, DNA repair, and play a role in various disorders such as cancer.

Restriction endonucleases occur ubiquitously among bacteria, archaea and in viruses of certain unicellular algae, and they are usually accompanied by a modification enzyme of identical specificity; together, the two activities form a restriction-modification (R-M) system- the prokaryotic equivalent of an immune system. More than 3,800 R-M enzymes have been characterized so far and they manifest 262 unique recognition specificities. These enzymes represent the largest family of functionally related enzymes. Based on the number and organization of subunits, cofactor requirements, catalytic mechanism, and sequence specificity, restriction enzymes have been classified into different types, Types I, II, III, and IV. R-M systems are important model systems for studying highly specific DNA-Protein interactions and serve as excellent systems for investigating structure-function relationship and for understanding the evolution of functionally similar enzymes with highly dissimilar sequence. In bacteria, DNA methyltransferases (MTases) associated with R-M systems protects the host DNA from cleavage by the cognate restriction endonuclease recognizing the same sequence and provides the integrity of host cell genome against foreign DNA invasion. The modification MTases catalyses the addition of a methyl group to one nucleotide in each strand of the recognition sequence using S-adenosyl-L-methionine (AdoMet) as the methyl group donor. Based on the chemistry of the methylation reaction catalyzed, DNA MTases are classified as C5 enzymes (endocyclic MTases), which transfer the methyl group to C5 position of cytosine, and N6 and N4 enzymes (exocyclic amino MTases), which transfer the methyl group to the exocyclic amino group of adenine or cytosine, respectively. DNA MTases of all three types contain conserved regions, which are responsible for catalysis and AdoMet binding, and variable regions known as target recognition domains (TRD), which determine the substrate specificity of a particular enzyme. Ten conserved amino acid motifs (I–X) are found in C5 MTases. Exocyclic DNA MTases are subdivided further into six groups (namely α, β, γ, ζ, δ and ε), according to the linear arrangements of three conserved motifs, the AdoMet-binding domain (FXGXG), the TRD (target recognition domain) and the catalytic domain (D/N/S)PP(Y/F). Base flipping has been proposed as a general mechanism used by all MTases in which the target base to be methylated is rotated 180º out of the DNA into a catalytic domain (motif IV). EcoP15I restriction enzyme (R.EcoP15I) belongs to the Type III restriction-modification (R-M) family. These enzymes are composed of two subunits, Res (Restriction) and Mod (Modification). The Mod subunit alone functions as a DNA methyltransferase in presence of AdoMet and magnesium and determines the specificity for restriction and methylation, whereas restriction activity requires the cooperation of both the Res and Mod subunits. EcoP15I methyltransferase (M.EcoP15I), a homodimeric enzyme catalyzes the transfer of a methyl group from AdoMet to the second adenine residue in the recognition sequence, 5’-CAGCAG-3’, in presence of magnesium ions. M.EcoP15I belongs to the β-subfamily of N6-adenine methyltransferases. In addition to the two highly conserved sequence motifs, FXGXG (motif 1) involved in AdoMet binding and DPPY (motif IV) involved in catalysis, the amino acid residues of the region 355-377 contains a PD(X)n(D/E)XK-like motif involved in metal binding. A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-Specific Endonuclease An interesting aspect of M.EcoP15I is that the methylation requires magnesium and magnesium binding to the PD(X)n(D/E)XK-like motif participates in base flipping. The PD-(D/E)XK superfamily of Mg2+-dependent nucleases were initially identified in structurally characterized Type II REases and later found in many enzymes involved in DNA replication, recombination and repair. The charged residues from the catalytic triads are implicated in metal ion mediated DNA cleavage. In EcoP15I DNA methyltransferase, a PD(X)n(D/E)XK like motif is present in which the partially conserved proline is replaced by methionine (MD(X)18(D/E)XK). Using site-directed mutagenesis methionine at 357 was changed to proline (M357P), which resulted in the formation of a Mg2+ binding/catalytic motif similar to several Mg2+-dependent endonucleases. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. The mutant protein specifically binds to the recognition sequence 5’-CAGCAG-3’ and cleaves DNA in presence of Mg2+. The engineered EcoP15I-M357P is an active, sequence-dependent restriction endonuclease that cleaves DNA 10/1 nucleotide away from its recognition sequence in the presence of Mg2+. Unlike the holoenzyme, R.EcoP15I, the engineered endonuclease neither requires AdoMet or ATP nor requires two sites in the inverted orientation for DNA cleavage. It is of potential interest to use such an engineered enzyme as a genetic manipulation tool. Dimerisation of EcoP15I DNA Methyltransferase is Required for Sequence Recognition and Catalysis In the cell, after each round of replication, substrate for any DNA MTase is hemimethylated DNA and therefore, only a single methylation event restores the fully methylated state. This is in agreement with the fact that most of the DNA MTases studied exist as monomers in solution. The peculiar feature of M.EcoP15I is that it methylates only one strand of the DNA, at the N6-position of the adenine residue. Earlier studies using gel filtration and glutaraldehyde cross-linking demonstrated that M.EcoP15I exists as dimer in solution. However, the significance of dimerisation in the reaction mechanism of EcoP15I MTase is not clear. Therefore, experiments have been performed to determine whether M.EcoP15I could function as a monomer and the significance of dimerisation, if any, in catalysis. Towards this a homology model of the M.EcoP15I was generated by “FRankenstein monster” approach. Residues D223, V225, and V392, the side chains of which are present in the putative dimerisation interface in the model were targeted for site-directed mutagenesis. These residues were mutated to lysine and their importance was studied. Methylation and in vitro restriction assays showed that the triple mutant was catalytically inactive. Interestingly, the mutations resulted in weakening of the interaction between the monomers leading to both monomeric and dimeric species. M.EcoP15I was inactive in the monomeric form and therefore, dimerisation might be the initial step in its function. This must be required for positioning of the target base of the DNA in the active-site pocket of the M.EcoP15I. A part of this interface may be involved in site-specific DNA binding. Dimerisation of M.EcoP15I is, therefore, a prerequisite for the high-affinity substrate binding needed for efficient catalysis. Understanding the role(s) of Amino and Carboxyl-terminal Domains of EcoP15I DNA Methyltransferase in DNA Recognition and Catalysis N-terminal and C- terminal domains (NTD and CTD) of proteins are known to play many important roles such as folding, stability, dimerisation, regulation of gene expression, enzyme activity and substrate binding. From the modeled dimeric structure of M.EcoP15I, it was hypothesized that N- and C-termini are in close proximity with each other. In addition, it was predicted that each monomer can bind to AdoMet and DNA. Towards understanding the role(s) of the N- and C-terminal domains of M.EcoP15I in its structure and function, N-, and C-terminal deletions were created. Interestingly, deletion of N-terminal 53 amino acids and C-terminal 127 amino acids from of EcoP15I MTase converted the dimeric enzyme to a stable, monomeric protein that was structurally stable but enzymatically inactive. Each monomer could bind single-stranded DNA but dimerisation was required for double-stranded DNA binding and methylation. This indicated that amino acids at the N- and C-termini are important for maintaining a proper dimeric structure for M.EcoP15I functions. Therefore, it can be proposed that in a complex three-dimensional structure, the NTD and CTD should be properly maintained in order to execute its function, including dimerisation and DNA binding. However, since the 3D structure of M.EcoP15I has not yet been determined, the biochemical, biophysical and bioinformatics approaches may serve to provide useful information on the relative contributions of the electrostatic forces and hydrophobic contacts to the structural stability. Understanding the structural organization and folding of M.EcoP15I is crucial to elucidation of the mechanism of action.

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No. of bitstreams: 1 poltronierioliveira_ab_me_sjrp.pdf: 1526886 bytes, checksum: b50424a42154b92cbde06c084bf0975d (MD5) Previous issue date: 2016-03-04 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introdução: A inflamação crônica desencadeada pela bactéria Helicobacter pylori (H. pylori), a qual é considerada o principal fator ambiental relacionado ao câncer gástrico, está associada ao desenvolvimento e progressão de lesões gástricas pré-cancerosas, desencadeando diversas modificações histológicas e moleculares que promovem a transformação maligna do estômago. Para isso, conta com fatores de virulência que promovem alterações superficiais e em vias de sinalização das células epiteliais gástricas. Consequentemente pode levar a alterações no padrão de expressão de genes supressores tumorais e da atividade de enzimas DNA metil transferases (DNMTs), responsáveis pela metilação do DNA e silenciamento gênico. Objetivos: O presente estudo avaliou se a infecção pela bactéria H. pylori, bem como sua erradicação, altera a expressão do RNAm dos genes supressores SOCS1, RPRM, RUNX3 e dos genes de DNMTs (DNMT1, DNMT3A e DNMT3B) em pacientes com gastrite crônica infectados (Hp+) em comparação com indivíduos com gastrite crônica sem infecção (Hp-). Além disso, investigou a ocorrência de correlação negativa entre a expressão do RNAm dos genes supressores tumorais com a dos genes das DNMTs, assim como a associação dos níveis de expressão gênica em relação aos fatores de risco idade, sexo, tabagismo, etilismo e genótipo bacteriano cagA. Material e Métodos: A quantificação relativa (RQ) do RNAm foi realizada por PCR (polymerase chain reaction) quantitativa em tempo real (qPCR) utilizando ensaios TaqMan® em 9 pacientes com gastrite crônica Hp- e 19 Hp+, sendo estes também avaliados três meses depois da terapia de erradicação bacteriana. O diagnóstico molecular e genotipagem do fator de virulência cagA foram realizados por PCR convencional. Resultados: Os resultados mostraram que a infecção pela H. pylori e sua erradicação não alteraram significantemente a expressão dos genes SOCS1, RPRM, RUNX3 e DNMTs, as quais apresentaram, de modo geral, expressão reduzida (RQ< 1,0), enquanto foi observado expressão mais elevada de SOCS1 e RPRM no grupo sem infecção Hp-. Quanto aos fatores de risco, também não foram encontradas associações significantes com os níveis de expressão dos genes avaliados. A análise de correlação não mostrou correlação negativa da expressão gênica entre os supressores tumorais e as DNMTs, mas evidenciou algumas correlações positivas entre a expressão dos genes SOCS1 e DNMT1 e do RPRM com DNMT3A e DNMT3B no grupo Hp+, que podem ter sido casuais. Conclusão: Nossos resultados não indicam que a infecção causada pela bactéria H. pylori e sua erradicação em pacientes com gastrite crônica afetam a expressão dos supressores tumorais SOCS1, RPRM, RUNX3 e das DNMTs, assim como que seja influenciada pelos fatores idade, sexo, tabagismo, etilismo e genótipo bacteriano cagA. Além disso, a expressão reduzida das DNMTs e ausência de correlação negativa com a dos genes supressores tumorais não permite indicar que a baixa expressão dos genes supressores tumorais seja devido a hipermetilação do DNA em consequência da infecção. / Introduction: Chronic inflammation caused by Helicobacter pylori (H. pylori), which is considered the main environmental factor related to gastric cancer, is associated with the development and progression of precancerous gastric lesions, triggering several histological and molecular changes that promote stomach malignant transformation. For this, it has virulence factors promoting superficial and signaling pathways of gastric epithelial cells changes. Consequently, it can lead to alterations in the expression of tumor suppressor genes and DNA enzyme activity methyl transferases (DNMTs), responsible for DNA methylation and gene silencing. Objectives: This study evaluated whether the infection by the bacterium H. pylori and its eradication change the mRNA expression of suppressor genes SOCS1, RPRM, RUNX3 and DNMTs (DNMT1, DNMT3A and DNMT3B) genes in patients with chronic gastritis infected (Hp+) compared to individuals with chronic gastritis without infection (Hp-). In addition, we investigated the occurrence of negative correlation between mRNA expression of tumor suppressor genes with the ones of DNMTs, as well as the association of gene expression levels in relation to the risk factors age, sex, smoking, drinking and bacterial genotype cagA. Methods: The relative quantification (RQ) mRNA was performed by PCR (polymerase chain reaction) quantitative real-time (qPCR) using TaqMan® assays in 9 patients with chronic gastritis Hp- and 19 Hp+, which are also evaluated three months after bacterial eradication therapy. The molecular diagnostics and genotyping of the virulence factors CagA were performed by standard PCR. Results: The results showed that the infection by H. pylori and eradication did not significantly alter the gene expression of SOCS1, RPRM, RUNX3 and DNMTs, which presented, in general, reduced expression (RQ <1.0); on the other hand, higher expression of SOCS1 and RPRM was observed in the group without Hp- infection. As for risk factors, no significant associations with the expression levels of evaluated genes were found. The correlation analysis not showed a negative correlation of gene expression in the tumor suppressor and DNMTs, but showed some positive correlations between the expression of SOCS1 and DNMT1 genes and RPRM with DNMT3A and DNMT3B the Hp + group, which may have been casual. Conclusion: Our findings do not indicate that the infection caused by the bacterium Helicobacter pylori and its eradication in patients with chronic gastritis affect the expression of tumor suppressor SOCS1, RPRM, RUNX3 and DNMTs, as it is influenced by factors such as age, sex, smoking, alcoholism and bacterial genotype cagA. Furthermore, the reduced expression of DNMTs and no negative correlation with the tumor suppressor genes do not indicate that the low expression of tumor suppressor genes is due to DNA hypermethylation in consequence of infection. / CNPq: 474.776/2013-1 / FAPESP: 2012/15036-8