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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - April 2022)
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
Diabetes is a chronic disease that results from the body’s inability to properly control circulating blood glucose levels. The loss of glucose homeostasis can arise either from a loss of β-cell mass because of immune-cell mediated attack, as in T1D, and/or from dysfunction of individual β-cells (in conjunction with target organ insulin resistance), as in T2D. Despite advances in current therapies to treat diabetes we are still far from a cure, and a greater understanding of the transcriptional pathways regulating islet development, function and survival will be critical if we are to achieve this goal. The aims of this dissertation were to delineate the role of the transcription factor Myt3 in β-cell function and survival. To this end we first examined the regulatory mechanisms involved in the control of Myt3 expression. We demonstrate that Myt3 expression is dependent on important islet transcription factors, including Foxa2, Pdx1 and Neurod1. We further established that Myt3 expression is regulated both developmentally, likely by the aforementioned factors, and by external stimuli including glucose and cytokines. From these early results we explored the effect of Myt3 suppression on the function and survival of β-cells. Our data show that reduced levels of Myt3 impair the ability of β-cells to migrate, which has potential implications for islet formation during development and compensatory islet neogenesis during diabetes progression, and leads to increased apoptosis. Lastly, to confirm these effects in vivo we studied the effects of Myt3 suppression in syngeneic islet transplants. Our data show that reduced Myt3 results in increased cell death in the grafts. Collectively, the data presented in this dissertation are an important step in clarifying the regulatory networks responsible for β-cell development, function and survival, and point to Myt3 as a potential therapeutic target for improving functional β-cell mass.
Master's Student Supervision (2010 - 2021)
Regulation of chromatin structure through posttranslational histone modifications is implicated in the induction of synaptic plasticity and memory formation. One such modification – histone H3 lysine 4 methylation (H3K4me) – has recently emerged as a key epigenetic modification necessary for consolidation of hippocampus-dependent memory. It is well-established that H3K4me levels across the genome are dynamically regulated by opposing activity of lysine methyltransferases (KMTs) and lysine demethylases (KDMs). They link dysregulation of H3K4 KMTs to neurodegenerative disorders, such as Alzheimer’s disease (AD). The major group of H3K4 KMTs in mammals are the Trithorax Group (TrxG) complexes, which can promote gene expression via distinct enzymatic (methylation of H3K4) and non-enzymatic (e.g. recruitment of other co-activators) mechanisms. In my project, I targeted the catalytic activity of TrxG complex and demonstrated that the loss of H3K4 methylation in mature hippocampal neurons leads to several intellectual abnormalities, such as the development of anxiety-like behaviour, recognition memory deficit, and impaired reversal memory with normal locomotory coordination in mice. Furthermore, I provided evidence of reduced H3K4 methylation in the hippocampus of AD by using a combination of patient samples and rodent disease model. Collectively, these results suggest that TrxG-mediated H3K4 methylation is required for a proper formation of hippocampal memory and may help shed light on H3K4 methylation as a novel therapeutic target for the treatment and prevention of AD.
The initial onset of type 1 diabetes, as well as islet graft rejection, is characterized by the autoimmune assault on the β-cells of the pancreatic islets of Langerhans. Resident and infiltrating immune cells secrete a cocktail of cytokines, such as IFNγ, Il-1β, and TNFα, which in turn, signal the β-cells to produce and secrete various chemokines and cytokines that lead to the recruitment of additional immune cells, eventually leading to β-cell failure and death. During these processes the expression of many genes becomes altered within β-cells, and we hypothesized that alterations to the chromatin states of β-cell cis-regulatory regions underlies these gene expression changes. The chromatin state of a given cis-regulatory region can be identified by the pattern of post-translational histone modifications on adjacent nucleosomes. For this study we focused on 4 histone modifications: Histone 3 Lysine 4 monomethylation (H3K4me1) and trimethylation (H3K4me3), Histone 3 Lysine 9 trimethylation (H3K9me3) and Histone 3 Lysine 27 trimethylation (H3K27me3); with a particular focus on H3K4me1 that is associated with active or poised enhancers and promoters. Our ChIP-Seq analysis revealed that, upon IFNγ, Il-1β, and TNFα exposure, many genomic regions in β-cells acquire de novo H3K4me1, despite being initially marked by the repressive histone modification H3K27me3. Many chemokine and cytokine genes were associated with these de novo enhancer regions, and the expression of many of these chemokine and cytokine genes is induced in islets exposed to IFNγ, Il-1β, and TNFα. We identified the Trithorax group (TrxG) complexes as likely candidates involved in the generation of these de novo enhancers, as they can contain proteins with H3K4 methyltransferase and H3K27me3 demethylase activity. To confirm the involvement of these complexes we attempted to block their activity by using an adenovirus expressing shRNAs targeting the core TrxG complex subunit Wdr5, and by using a small molecule selective inhibitor (GSK-J4) of the H3K27me demethylases Utx and Jmjd3. Both approaches resulted in blunting of the IFNγ, Il-1β, and TNFα induced expression of proinflammatory cytokines, with GSK-J4 having a more pronounced effect.