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

Transcription-associated RNA surveillance is vital to the well-being of the cell by processing both coding and noncoding RNA (ncRNA). When left unregulated, RNA transcripts can cause genomic instability by forming structures called R-loops that leave a single strand of DNA exposed to potentially harmful events like nicks and mutations. The RNA exosome is the 3’ exoribonuclease that plays an important role in degrading both coding and ncRNA, and its role in processing ncRNA transcripts has been demonstrated extensively in the recent past. The role of ncRNA transcription and the RNA exosome is best exemplified in B cell programmed DNA recombination, namely class switch recombination (CSR). In CSR, ncRNA transcription and RNA exosome recruitment to switch sequences allows for targeting of the activation-induced cytidine deaminase (AID) enzyme and ultimate formation of DNA double strand breaks, which results in recombination between the appropriate switch regions. While the mechanism of the RNA exosome in processing this transcribed ncRNA and regulating CSR is well-established, the mechanism of RNA exosome regulation and recruitment remains unknown. RNA base modifications are another facet of RNA surveillance that can have profound effects on RNA biology. While some modifications are fixed, reversible base modifications such as methylation are particularly influential due to their dynamic nature. Among the most widely studied of reversible modifications is that of N⁶-methyladenosine (m⁶A). M⁶A has been identified for its role in regulation of gene expression and cell differentiation via its effect on regulating mRNA levels and downstream function. Although it has been shown to be present on some ncRNA, the role of m6A in regulating ncRNA turnover and genome dynamics is largely unclear. We interrogated the hypothesis that m6A plays a role in regulating RNA exosome recruitment in B cell programmed DNA recombination. We initially assessed the role of the RNA exosome cofactor MPP6 in CSR using CRISPR/Cas9-mediated knockout mouse B cell hybridoma CH12F3 cells (CH12 cells) and established a connection between the RNA exosome and m6A by demonstrating an interaction between MPP6 and m6A reader protein YTHDC1. We then utilized mouse models to knockout METTL3, a m6A methyltransferase, in primary B cells and assessed the efficiency of CSR. We performed RNA sequencing to assess the levels of ncRNA in B cells upon METTL3 loss and find that primarily G-rich RNA transcripts are accumulated upon METTL3 loss, exemplified by switch region transcript SμGLT. In order to specifically show that methylation of SμGLT is required for efficient CSR, we used fusion proteins of catalytically dead Cas13b fused to either m6A demethylase ALKBH5 or FTO and targeted SμGLT with a guide RNA specific for the transcript. We found that methylation of SμGLT specifically is required for efficient CSR, with targeted demethylation resulting in a decrease in CSR efficiency. We also interrogated the role of m6A reader protein YTHDC1 in CSR via CRISPR/Cas9-mediated knockout CH12 cells to assess the mechanism of m6A-mediated downstream function. We found that YTHDC1 is also required for efficient CSR, likely via interaction with MPP6 and the RNA exosome to degrade SμGLT. We also assessed the role of MPP6, METTL3, and YTHDC1 in facilitating the association between AID and the RNA exosome via 3D stochastic optical reconstruction microscopy (3D-STORM) and found that all three components are required for nucleation of AID and the RNA exosome (for which helicase MTR4 was used as a proxy). We then wanted to assess whether m⁶A loss promotes genomic instability in B cell programmed DNA breaks. We performed linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) to assess the levels of on- versus off-target rearrangements in METTL3 knockout primary B cells. We found that upon METTL3 loss there is an increase in off-target, non-immunoglobulin translocations as well as an increase in microhomology-mediated end joining, indicative of defects in on-target recombination and the canonical nonhomologous end joining pathway. These data suggest that m⁶A is required for targeted programmed DNA rearrangements and protection of nonhomologous end joining in B cells. Taken together, these findings have elucidated a novel mechanism by which m6A regulates ncRNA turnover, RNA exosome targeting, and genome dynamics in B cell programmed DNA rearrangements.

Schizophrenia is a disabling psychiatric and neurodevelopmental disorder that represents a tremendous public health burden. Despite the inroads made in the treatment of its symptoms, understanding its etiology and pathophysiology remains challenging due to the genetic heterogeneity of the disease and the corresponding complexity of the neural systems which it affects. In recent years, the development of next generation sequencing and substantial progress in the field of psychiatric genetics have revealed the important role of individually rare but collectively common heritable and de novo mutations (DNMs) in the complex genetic architecture of schizophrenia. Previously, we had identified SETD1A encoding a histone methyltransferase, as a high-risk gene for schizophrenia, which has been confirmed extensively through follow-up meta-analyses. This discovery emphasized the important role that neural gene regulation plays in the coordination of complex cognitive processes. However, it is unclear how to translate a ubiquitous molecular process such as chromatin modification into a mechanistic and disease-specific insight. Our previous comprehensive analysis of mutant mice carrying a loss of function (LoF) allele in the Setd1a orthologue uncovered the role of SETD1A in gene regulation, neuronal architecture, synaptic plasticity, neuronal ensemble activity and cognitive function and showed that neurocognitive deficits that derive from Setd1a deficiency can be reversed by pharmacological interventions during adulthood. Our previous ChIP-Seq analysis showed a striking overlap between SETD1A, MEF2, and LSD1 targets at enhancers in the prefrontal cortex (PFC). Since MEF2 is an activity dependent transcription factor, we hypothesized that SETD1A may also modulate activity-dependent gene expression in the brain. To elucidate the effects of Setd1a deficiency on activity-dependent transcription, we established an in vitro neuronal activity dependent gene (ADG) expression assay and identified genes modulated by neuronal activity using ChIP-Seq and RNA-Seq assays. We found a remarkable overlap of a dynamic pattern of activity-dependent recruitment of SETD1A, LSD1 and MEF2 to enhancers of ADGs. Our results showed Setd1a deficiency affects transcription in an activity-dependent manner and transcriptional alteration induced by Setd1a deficiency under neuronal activation can be attenuated by inhibition of LSD1 activity. In addition, we investigated how SETD1A modulates MEF2 transactivation activity by performing luciferase assays. Our results suggest that SETD1A represses MEF2 activity but the repression is unlikely to be mediated by lysine methylation. We also performed behavioral analyses of Setd1a+/- mice and found that the social behavior and social memory were impaired in female Setd1a+/- mice but remained intact in male Setd1a+/- mice. Ultimately, future work is underway to analyze the targets of SETD1A, which in turn could lead to the development of therapeutic strategies to reverse the progression of schizophrenia.

Indiana University-Purdue University Indianapolis (IUPUI) / Setd1 histone 3 lysine 4 methyltransferases are critical for epigenetic regulation and gene expression. Setd1a is multiprotein complex comprised of several critical subunits including wdr82, which is essential for embryonic development, and cfp1, critical for regulation of both activation and repression of transcriptional programs required in basic and developmental cellular processes.

In the post-genomic era, one of the most important goals for the community of plant biologists is to take full advantage of the knowledge generated by the Arabidopsis thaliana genome project, and to employ state-of-the-art functional genomics techniques to assign function to each gene. This will be achieved through a complete understanding of what all cellular components do, and how they interact with one another to produce a phenotype. Among the proteins encoded by the Arabidopsis genome are 24 related carboxyl methyltransferases that belong to the SABATH family. Several of the SABATH methyltransferases convert plant hormones, like jasmonic acid, indole-3-acetic acid, salicylic acid, gibberellins, and other plant constituents into methyl esters, thereby regulating the biological activity of these molecules and, consequently, myriad important physiological processes. Our research aims to decipher the function of proteins belonging to the SABATH family by applying a combination of genomics tools, including genome-wide expression analysis and gas-chromatography coupled with mass spectrometry-based metabolite profiling. Our results, combined with available biochemical information, provide a better understanding of the physiological role of SABATH methyltransferases, further insights into secondary plant metabolism and deeper knowledge of the consequences of modulating the expression of SABATH methyltransferases, both at the genome-wide expression and metabolite levels. / Ph. D.

<div>Protein N-terminal methyltransferases (NTMTs) are a family of enzymes that methylate the α-N-terminus of a variety of protein substrates. Both NTMT1 and NTMT2 recognize a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) to install 1-3 methyl group(s) on the substrates. NTMT1 plays important roles in mitosis regulation, chromatin interactions, and DNA damage repair. Another member NTMT2 shares ~50% sequence similarity and the same substrate recognition motif although NTMT2 was initially characterized as a mono-methyltransferase. To understand the molecular mechanism of NTMT2, we obtained the first co-crystal structure of NTMT2 in complex with its peptide substrate. After an extensive investigation of substrate recognition and methylated products of NTMT1/2, we found out that NTMT2 can fully methylate G/P-PKRIA peptides despite a predominant mono-methyltransferase. Moreover, we identified a gatekeeper N89 in NTMT2 that controls the substrate entry and the product specificity of NTMT2.</div><div>To elucidate the biological functions of NTMT1/2-catalyzed N-terminal methylation, we applied two different strategies to discover cell-potent inhibitors. Guided by the co-crystal structures of NTMT1 in complex with previously reported inhibitors, we designed and synthesized a series of new peptidomimetic inhibitors. By introducing more hydrophobic groups, the most cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM) exhibited over 2-fold increased inhibition on cellular N-terminal methylation levels with an IC50 value of ~50 µM compared to previously reported peptidomimetic inhibitor DC541. Meanwhile, we also discovered the first potent small molecule inhibitor Genz-682452 (IC50 = 0.5 ± 0.04 µM) after screening ~58,000 compounds. Subsequent structural modifications led to the discovery of GD433 (IC50 = 27 ± 0.5 nM) with a 20-fold increased potency compared to the initial hit Genz-682452. Inhibition mechanism indicated both inhibitors bind to peptide-binding pocket and co-crystal structures of both Genz-682452 and GD433 with NTMT1 confirmed their binding modes. Furthermore, GD433 shows over 7-fold selectivity over other major 40 protein methyltransferases and DNA methyltransferase and exhibits improved selectivity for NTMT1 over glucosylceramide synthase (GCS). GD433 significantly decreases the cellular N-terminal methylation level of NTMT1 substrates RCC1 and SET at 10 nM in both HEK293 and HCT116 cells, providing a valuable probe for cell-based studies in the future.<br></div><p><br></p>

Within the past 70 years since the discovery of 5-methylcytosine, we have acquired considerable knowledge about genomic DNA methylation patterns, the dynamics of DNA methylation throughout development, and the enzymatic machinery that establishes and perpetuates genomic methylation patterns. Nonetheless, in the field of epigenetics major questions remain open about the mechanisms of spatiotemporal control that exist to ensure the fidelity of methylation patterns. This thesis aims to decipher the regulatory logic and upstream pathways influencing one of the DNA methyltransferases by leveraging the diverse resources of molecular genetics, biochemistry, and structural biology. The primary subject of my research, DNA methyltransferase 1 (DNMT1), is crucial for maintaining genomic methylation patterns upon DNA replication and cell division. In addition to its C-terminal catalytic domain, mammalian DNMT1 harbors several N-terminal domains of unknown function: a succession of seven glycine-lysine (GK) repeats, resembling histone tails, and two Bromo-Adjacent Homology (BAH) domains that are absent from bacterial DNA methyltransferases. The work I present in this thesis characterizes the role of these hitherto enigmatic domains in regulating DNMT1 activity. In my studies, I found that mutation of the (GK) repeats motif leads to de novo methylation by DNMT1 specifically at paternally imprinted genes. Conventionally, de novo methylation is thought to be undertaken by complete different enzymes, DNMT3A and DNMT3B, whereas DNMT1 is limited to perpetuating the patterns these other methyltransferases had set down. Recombinant DNMT1 had been previously shown to efficiently methylate unmethylated DNA substrate in vitro, but this is the first time its de novo methyltransferase capability has been observed in vivo. Based on these data, I propose a new model in which DNMT1 is the enzyme responsible for laying down de novo methylation patterns at paternally imprinted genes in the male germline, explaining the previously observed non-essential role of other DNA methyltransferases in the establishment of paternal imprints. Furthermore, I demonstrated that acetylation of the (GK) repeats motif inhibits this de novo methyltransferase activity of DNMT1, making this particular motif an essential regulatory platform for controlling the diverse in vivo functions of the enzyme. Though the (GK) repeats motif had previously been proposed to regulate the stability of DNMT1 protein through its interaction with the deubiquitinase USP7, I tested the biological relevance of this interaction and found that USP7 deletion does not alter DNMT1 protein levels. In fact, USP7 appears to play no part in regulating maintenance DNA methylation, as I present evidence that USP7 localization to replication foci is entirely independent of DNMT1. Finally, I demonstrated that the tandem BAH domains of DNMT1 are required for its maintenance methyltransferase activity as they are involved in targeting the enzyme to replication foci during S phase. Based on biochemical data supporting an interaction between DNMT1's BAH1 domain and histones, I propose that this targeting could occur through BAH1's recognition of specific histone modifications, thus providing a potential mechanistic link between maintenance DNA methylation and chromatin markings. This thesis identifies DNMT1 as a novel de novo methyltransferase in vivo and also characterizes the regulatory functions of the enzyme's BAH domains and the (GK) repeats. These results elucidate the multiple regulatory mechanisms within the DNMT1 molecule itself that control its functions in mammalian cells, thereby providing critical insights as to how the DNA methylation landscape takes shape and yielding surprising revelations about the parts that well-studied proteins have to play in this process.

Protein arginine methyltransferase 1 (PRMT1) is a key posttranslational modification enzyme that catalyzes the methylation of specific arginine residues in histone and nonhistone protein substrates, regulating diverse cellular processes such as transcriptional initiation, RNA splicing, DNA repair, and signal transduction. Recently the essential roles of PRMT1 in cancer and cardiovascular complications have intrigued much attention. Developing effective PRMT inhibitors therefore is of significant therapeutic value. The research on PRMT inhibitor development however is greatly hindered by poor understanding of the biochemical basis of protein arginine methylation and lack of effective assays for PRMT1 inhibitor screening. Herein, we report our effort in the kinetic mechanism study as well as the fluorescent probe and inhibitor development for PRMT1. New fluorescent reporters were designed and applied to perform single-step analysis of substrate binding and methylation of PRMT1. Using these reporters, we performed transient-state fluorescence measurements to dissect the rate constants along the PRMT1 catalytic coordinate. The data give evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, we identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. On the other hand, we discovered a type of naphthyl-sulfo (NS) compounds that block PRMT1- mediated arginine methylation at micromolar potency through a unique mechanism: they directly target the substrates but not PRMT enzymes for the observed inhibition. We also found that suramin, an anti-parasite and anti-cancer drug bearing similar functional groups, effectively inhibited PRMT1 mediated methylation. These findings about novel PRMT inhibitors and their unique inhibition mechanism provide a new way for chemical regulation of protein arginine methylation. Addionally, to dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. While certain acetylations present a repressive impact on PRMT-1 mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases activity of PRMT1 but increases that of PRMT5. Furthermore, hyperacetylation increases the content of ordered secondary structures of H4 tail. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation, and unravel that complex intercommunications exist between different posttranslational marks in cis.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 120-130).