<p>DNA methylation is an epigenetic modification that is nearly ubiquitous. Eukaryotic DNA methylation contributes to the regulation of gene expression and maintaining genome integrity. In mammals, DNA methylation occurs primarily on the C5 carbon of cytosine in a CpG dinucleotide context and is catalyzed by the DNA methyltransferases, DNMT1, DNMT3A and DNMT3B. While <i>dnmt3a</i> and <i>dnmt3b</i> genes are highly homologous, the enzymes have distinct functions. Some previous reports suggested differences in the enzymatic behavior of DNMT3A and 3B, which could affect their biological roles. The goal of my thesis work was to characterize kinetics mechanisms of DNMT3A and 3B, and to identify the similarities and differences in their catalytic properties that contribute to their distinct biological functions. Given the sequence similarity between the enzymes, we asked whether DNMT3B was kinetically similar to DNMT3A. In a series of experiments designed to distinguish between various kinetics mechanisms, we reported that unlike DNMT3A, DNMT3B methylated tandem CpG on DNA in a processive manner. We also reported that the disruption of the R-D interface, critical for the cooperativity of DNMT3A, had no effect on DNMT3B activity, supporting the non-cooperative mechanism of this enzyme. </p> <p>DNMT3A is frequently mutated in numerous cancers. Acute Myeloid Leukemia (AML) is a malignancy of hematopoietic stem cells in which numerous patients exhibit a high frequency of the heterozygous somatic mutation Arg882His in DNMT3A. Through thorough consensus motif building, we discovered a strong similarity in CpG flanking sequence preference between DNMT3A Arg882His variant and DNMT3B enzyme. Moreover, we found that the variant enzyme has the same kinetics mechanism as DNMT3B, indicating a gain-of-function effect caused by the mutation. This change is significant because the variant enzyme can aberrantly methylate DNMT3B targets in AML cells and effect global gene expression. In particular, given that DNMT3B has been shown to have oncogenic properties, this suggests that the Arg882His variant can acquire similar oncogenic properties and drive AML development.</p> <p>Taken together, my thesis work provides novel insights into the relationship between the biochemical properties and the biological functions of DNMT3A and 3B. </p>

<p dir="ltr">The BRG1/BRM-associated factor (BAF) complexes, also called SWI/SNF, are multi-subunit chromatin remodelers that regulate chromatin compaction in an ATP-dependent manner. In the past decade, BAF complexes have been under the spotlight in cancer research, especially after proteomic analyses revealed the genes encoding the subunits are amongst the most frequently mutated genes in cancer. The present dissertation focuses on prostate cancer (PCa), a disease in which the role of the BAF subunits is increasingly being explored but is yet to be defined as a potential therapeutic target. According to the GLOBOCAN report, PCa is the second most frequent cancer in males worldwide. Since most of the variants of PCa rely on the androgen receptor (AR) axis, surgical or chemical castration and androgen deprivation therapy (ADT) are the main treatment strategies for PCa patients. Even though these therapeutic approaches prolong survival, reduce tumor burden, and relieve symptoms, PCa patients eventually relapse and develop castration resistant PCa (CRPC). At present, the mechanisms underlying ADT resistance are not fully understood, current efforts focus on finding new targets for PCa treatment.</p><p dir="ltr">In the projects included in this dissertation we explored the function of the PBAF complex, a BAF subtype, in a variety of models of PCa and its potential as a therapeutic target by inhibiting or depleting its different subunits. To do so we (i) developed the first inhibitors for BRD7 (a subunit unique to PBAF) and (ii) established cell-based assays in multiple PCa cell lines to study BRD7 and other PBAF unique subunits.</p><p dir="ltr">Bromodomain-containing proteins are readers of acetylated lysine and play important roles in cancer. Bromodomain-containing protein 7 (BRD7) has been implicated in multiple malignancies; however, there are no selective chemical probes to study its function in disease. Using crystal structures of BRD7 and BRD9 bromodomains (BDs) bound to BRD9-selective ligands, we identified a binding pocket exclusive to BRD7. We synthesized a series of ligands designed to occupy this binding region and identified two inhibitors with increased selectivity towards BRD7, 1-78 and 2-77, which bind with submicromolar affinity to the BRD7 BD. Our binding mode analyses indicate that these ligands occupy a uniquely accessible binding cleft in BRD7 and maintain key interactions with the asparagine and tyrosine residues critical for acetylated lysine binding. Finally, we validated the utility and selectivity of the compounds in cell-based models of prostate cancer.</p><p dir="ltr">There are three BAF complexes that have been biochemically characterized up to date: canonical BAF (cBAF), polybromo-associated BAF (PBAF) and GLTSCR1/like-containing BAF (GBAF or ncBAF). All BAF complexes are characterized by containing an ATPase and accessory subunits that may be shared between them or unique to each subtype. PBAF, the BAF subtype of interest of this dissertation, contains four unique subunits: BRD7, PBRM1, ARID2 and BAF45A. We showed that knocking down BRD7 and ARID2 leads to reduction of cell viability in PCa cells with ligand-dependent and independent AR signaling, while knocking down PBRM1 leads to reduction in viability of cells with only ligand-dependent AR signaling. We also performed a chromatin immunoprecipitation assay with BAF45A and observed that it does not colocalize with AR binding sites, indicating that the mechanism by which PBAF regulates AR signaling is indirect. This observation was further supported by the fact that knocking down BRD7 prevents expression of genes related to adaptive processes, but not AR target genes, in response to androgen treatment. Further mechanistic studies will aid in understanding the function of PBAF in PCa. However, overall, our results indicate that PBAF is a promising therapeutic target in PCa models expressing AR, including CRPC systems.</p>