Characterizing the interaction between Inhibitor of Growth (ING) proteins and the nucleosome




Williamson, Bradley

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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.



chromatin, epigenetics, inhibitor of growth proteins, tumour suppresors, nucleosome, protein binding, transcription