Bradford Hoffman
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Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Aims:Insufficient insulin release by β-cells is the primary etiology in type 2 diabetes and coincides with impaired expression of genes essential for β-cell function, but drivers of gene expression dysregulation are not well resolved. Alterations to the genome-wide enrichment and organization of chromatin post-translational modifications may promote gene expression dysregulation. Here, I investigate the role of H3K4me3 in mature β-cells and how its organization in chromatin is linked to the unique β-cell gene transcriptome in health and diabetes. I further test how its enrichment is altered by external challenges in the form of type 2 diabetes-like stresses or perturbation of one carbon metabolism.Methods:To study the functional importance of H3K4me3 in mature β-cells, we depleted H3K4me3 in β-cells of mature mice using an inducible Dpy30 deletion model under control of the Pdx1 or Ins1 promoter and performed a panel of metabolic, transcriptomic, and epigenetic tests. We compared H3K4me3 enrichment patterns with gene expression changes that occur in islets in a mouse model of type 2 diabetes and in human type 2 diabetes. We then examined the metabolic and transcriptomic consequences of folic acid restriction in mouse islets.Results:H3K4me3 contributes to gene expression in mature β-cells. H3K4me3 contributes to H3K27ac levels and, in the absence of H3K4me3, promoter-associated H3K4me1 is partially sufficient to maintain expression. H3K4me3 peak breadth is correlated with gene expression dysregulation in type 2 diabetes in mice and humans. Using a genetic mouse model to impair the methyltransferase activity of trithorax group complexes, we find that reduction of H3K4me3 reduces insulin production and glucose-responsiveness and increases transcriptional entropy. H3K4me3 in mouse β-cells is particularly required for the expression of genes that are dysregulated in a mouse model of type 2 diabetes. While locus-specific alterations are observed, global enrichment of H3K4me3 in islets is robust against external disruption of glucose homeostasis and one-carbon metabolism.Conclusions/interpretation:Overall, this thesis shows that H3K4me3 contributes to expression of genes essential for β-cell identity and function in mature β-cells and implicates dysregulation of H3K4me3 as a factor contributing to β-cell dysfunction in type 2 diabetes by altering gene expression patterns.
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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
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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.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
Background: The deposition and removal of histone modifications are dictated by the intracellular levels of metabolites produced during cellular metabolism. Metabolites of interest include; S-adenosylmethionine (SAM), the key methyl donor for histone methylation; and S-adenosylhomocysteine (SAH), produced following methyl donation. Histone modifications, in turn, regulate gene expression. Dysregulation of β-cell metabolism in type 2 diabetes (T2D) may impact the levels of key metabolites, resulting in pathological alterations of chromatin state and gene expression. I hypothesize that β-cells from a diet-induced mouse model of T2D will have altered levels of key metabolites required as co-factors for chromatin-modifying enzymes, which will alter histone modifications at key β-cell gene loci and produce a pathological gene expression pattern.Methods: Using a diet-induced mouse model of T2D (western diet, WD) I performed transcriptomic and epigenetic analysis in β-cells from male and female mice fed a WD or a control diet (CD). I compared enrichment and combinational patterns of histone modifications across the β-cell genome and at differentially expressed genes (DEGs). I also examined SAM and SAH concentrations in islets. Results: Contrary to my hypothesis, islet SAM and SAH concentrations were not different between male and female WD and CD mice. The β-cell transcriptomes of WD mice had multiple DEGs to control mice. Expression of genes associated with oxidative phosphorylation were lower in WD mice. Genes associated with histone demethylation were upregulated in WD females, but not observed in WD males. Levels of H3K4me1 were greater, and H3K4me3 and H3K27ac/me3 were lower in WD mice. Based on the combination of the four histone modifications, chromatin states were annotated at the promoters of the DEGs and compared between WD and CD mice. A positive relationship between chromatin state and gene expression was identified, with higher active chromatin states at promoters of genes with higher expression, and lower active chromatin states at promoters of genes with lower expression in WD mice compared to control mice. Conclusion: The histone modification landscapes within β-cells were dramatically changed in a diet-induced mouse model of T2D and are related to gene expression profiles but are unrelated to SAM and SAH concentrations.
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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.
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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.
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