Inhibitor of growth (ING) proteins have been classified as type II tumour suppressor proteins due to their ability to facilitate cellular events such as chromatin remodelling, apoptosis, angiogenesis, DNA replication, DNA repair, cell cycle progression, cell senescence and hormone response regulation. These processes are all associated with combating oncogenesis; conversely, recent evidence suggesting that ING proteins also function as oncogenes in certain cancers has spurred the investigation of ING proteins as potential anticancer targets. In order to better understand the complex role ING proteins play in the cell, the mechanisms that direct ING proteins to the chromatin template require extensive study. This dissertation investigates the role the chromatin environment plays in recruiting ING proteins by characterizing the interaction between ING proteins and chromatin. ING proteins have been shown to interact with the histone H3 lysine 4 trimethylated (H3K4me3) epigenetic mark through binding studies between peptides comprising the ING plant homeodomain (PHD) finger and the H3 N-terminal tail. However, these studies do not take into account the effect of organizing H3 into a nucleosome or the effect of the remaining ING protein structural domains. In order to address these elements, this dissertation describes binding studies between the PHD finger of Yng1 (Yng1PHD) and H3K4me3 in the context of a nucleosome, and between full-length Xenopus laevis ING1 (xING1) and H3K4me3 in the context of a nucleosome. A 6XHis tagged xING1 protein was purified, Yng1PHD was obtained from Dr. Leanne Howe, and an analog of H3K4me3 (H3KC4me3) was installed into recombinant H3 protein and used to reconstitute nucleosomes. Affinity-tag based anti-Yng1PHD and anti-xING1 pull-down assays were then used to display an in vitro H3K4 methylation-dependent interaction between Yng1PHD / xING1 and H3KC4me3 containing nucleosomes. In addition, analytical ultracentrifuge (AUC) analysis of the xING1 protein displayed the presence of 3 species containing sedimentation coefficients consistent with those that would be expected from monomeric, dimeric and tetrameric forms of xING1. Several studies have focused on the interaction between ING proteins and DNA binding proteins such as transcription factors and hormone receptors which recruit ING proteins to specific genes. However, little knowledge is available regarding the role chromatin plays in recruiting ING proteins with the exception of the interaction between the ING PHD fingers and H3K4me3. This dissertation addresses this gap in knowledge by investigating the nature of chromatin bound by the human ING1b (hING1b) protein. For this purpose, HEK293 cells were transfected with a Flag-hING1b construct. Upon fractionation of the HEK293 chromatin, Flag-hING1b was found to localize exclusively to the “Pellet” fraction. ChIP analysis of the HEK293 chromatin showed that Flag-hING1b bound nucleosomes were deprived of H3K9me3, H3K27me3 and H3S10P, contained no enrichment for H3K4me3 and H3K36me3, and were significantly enriched for H2A.Z. Lastly, a hING1b-GFP construct was transiently transfected into SKN-SH human neuroblastoma cells and found to be evenly distributed throughout the nucleus with moderate enrichment on chromatin and within the nucleolus. / Graduate

Eukaryotic organisms require the ability to respond to their environments. They do so by utilizing signal transduction pathways that allow for signals to effect final biological responses. Many times, these final responses require new gene expression events that have been stimulated or repressed within the nucleus. Thus, much of the understanding of signal transduction pathways converges on the understanding of how signaling affects gene expression alterations (Kumar et al., 2004). The regulation of gene expression involves the modification of chromatin between condensed (closed, silent) and expanded (open, active) states. Histone modifications, such as acetylation, can determine the open versus closed status of chromatin. The PHD (Plant HomeoDomain) finger is a structural domain primarily found in nuclear proteins across eukaryotes. This domain specifically recognizes the epigenetic marks H3K4me2 and H3K4me3, which are di- and tri-methylated lysine 4 residues of Histone H3 (Loewith et al., 2000; Kuzmichev et al., 2002; Vieyra et al. 2002; Shiseki et al., 2003; Pedeux et al., 2005, Doyon et al., 2006). It is estimated that there are ~150 proteins that contain the PHD finger in humans (Solimon and Riabowol, 2007). The PHD finger is conserved in yeast and plants, however an analysis of this domain has only been performed done in Arabidopsis thaliana (Lee et al., 2009). The work presented in this report aims to extend the analysis of this domain in plants by identifying the PHD fingers of the crop species Oryza sativa (rice). In addition, a phylogenetic analysis of all PHD fingers in Arabidopsis and rice was undertaken. From these analyses, it was determined that there are 78 PHD fingers in Arabidopsis and 70 in rice. In addition, these domains can be categorized into classes and groups by defining features within the conserved motif. In a separate study, I investigated the function of two of the PHD finger proteins from Arabidopsis, ING1 (INhibitor of Growth1) and ING2. In humans, these proteins can be found in complexes associated with both open and closed chromatin. They facilitate chromatin remodeling by recruiting histone acetyltransferases and histone deacetylases to chromatin (Doyon et al., 2006, Pena et al., 2006). In addition, these proteins recognize H3K4me2/3 marks and are believed to be "interpreters" of the histone code (Pena et al., 2006, Shi et al., 2006). To understand the function of ING proteins in plants, I took a reverse genetics approach and characterized ing1 and ing2 mutants. My analysis revealed that these mutants are altered in time of flowering, as well as their response to nutrient and stress conditions. Lastly, I was able to show that ING2 protein interacts in vitro with SnRK1.1, a nutrient/stress sensor (Baena-Gonzalez et al., 2007). These results indicate a novel function for PHD proteins in plant growth, development and stress response. / Master of Science