The post-translational modifications (PTMs) of histones regulate many cellular processes including transcription, replication, DNA repair, recombination, and chromosome segregation. A large number of combinations of PTMs are possible, with methylation being one of the most complex, since it is found in three states and is recognized in a sequence specific context. Methylation of histones at key lysine residues has been shown to work in concert with other modifications to provide a Histone Code that may determine heritable transcriptional conditions in normal and disease states. On the most basic level it is pivotal to understand how and by which proteins the numerous PTMs are recognized, as well as mechanisms for downstream signal propagation. To address this need we developed a high-throughput method that allows analysis of up to 600 PTMs in a single experiment. This approach was utilized to characterize macromolecules interacting with the specific modifications on histone tails and to screen for the marks that bound to Malignant Brain Tumor (MBT) proteins, important chromatin regulators implicated in cancer. All MBTs recognized either mono- or dimethyllysine histone marks, and using structure-based mutants we identified a triad of residues that were responsible for this discrimination. These results provide the foundation for the rational design of highly selective MBT inhibitors. Additionally, this thesis describes combinatorial recognition of histone modifications, as proposed in the original Histone Code hypothesis. We demonstrate that Tudor domains of UHRF1, a protein involved in epigenetic maintenance of DNA methylation, is able to read a dual modification state of histone H3 in which it is trimethylated at lysine 9 and unmodified at lysine 4. This study provides an elegant example of the combinatorial readout of histone modification states by a single domain. Together, our findings offer mechanistic insights into the recognition of methylated histone tails by MBT domains and Royal Family in general.

Genomic imprinting refers to the parent-of-origin specific monoallelic expression of a gene. Imprinted genes are often clustered in the genome and their expression is regulated by an imprinting centre (IC). ICs are regions of DNA that propagate the parental specific regulation of gene expression, which are usually characterized by differential DNA methylation, histone marks and the presence of non-coding RNAs. Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with the dysregulation of imprinted gene expression on human chromosome band 11p15.5. The 11p15.5 imprinted region has two imprinting centres, IC1 and IC2. IC1 is telomeric and regulates the imprinted expression of the genes H19 and IGF2. IC2 is ~700kb centromeric and is associated with a cluster of nine imprinted genes including CDKN1C, KCNQ1 and an imprinted non-coding RNA associated with IC2, KCNQ1OT1. Loss of differential DNA methylation at IC2 is seen in 50% of patients with BWS with loss of imprint of the non-coding RNA KCNQ1OT1 and associated with a decreased expression of the putative tumour suppressor CDKN1C. Patients with BWS also have a thousand-fold increased risk of pediatric cancer. The focus of this thesis involves investigation of dysregulation of imprinting in three groups of BWS patients. Firstly, I show that BWS patients with alveolar rhabdomyosarcoma have constitutional loss of methylation at IC2 and biallelic expression of KCNQ1OT1. Secondly, loss of methylation at IC2 has been previously associated with female monozygotic twins discordant for BWS. In male monozygotic twins with BWS, however, the molecular lesions reflect the molecular heterogeneity seen in BWS singletons. Thirdly, BWS patients associated with translocations and inversions that have breakpoints within the KCNQ1 gene near IC2 show regional gain of DNA methylation around the breakpoint and decreased expression of CDKN1C. Therefore, using a rare collection of BWS patients, I have attempted to determine the various roles of the imprinting centres IC1 and IC2 and their involvement in tumourigenesis, monozygotic twinning and structural chromosomal rearrangements causing BWS.

Acute myeloid leukemia (AML) is a genetic disease whereby sequential genetic aberrations alter essential white blood cell development leading to differentiation arrest and hyperproliferation. Pertinent animal models serve as essential intermediaries between in vitro molecular studies and the use of new agents in clinical trials. We previously generated a transgenic zebrafish model expressing human NUP98-HOXA9 (NHA9), a fusion oncogene found in high-risk AML. This expression yields a pre-leukemic state in both embryos and adults. Using this model, we have identified the overexpression of dnmt1 and the Wnt/β-catenin pathway as downstream contributors to the myeloproliferative phenotype. Targeted dnmt1 morpholino knockdown and pharmacological inhibition with methyltransferase inhibitors rescues NHA9 embryos. Similarly, inhibition of β-catenin with COX inhibitors partially restores normal hematopoiesis. Interestingly, concurrent treatment with a histone deacetylase inhibitor and either a methyltransferase inhibitor or a COX inhibitor, synergistically inhibits the effects of NHA9 on embryonic hematopoiesis. Thus, we have identified potential pharmacological targets in NHA9-induced myeloid disease that may offer a highly efficient therapy with limited toxicity – addressing a major long-term goal of AML research.

<p>Herein we detail the progress made at understanding the overall process of CD8<super>+</super> T-lymphocyte noncytolytic antiviral response (CNAR). This response is comprised of 3 key components, the virus, the effector cell and the target cell, each of which contribute to noncytolytic suppression. During the course of CNAR, the effector cells express antiviral factors that induce intracellular events in the target cell resulting in host-pathogen interactions that inhibit HIV-1 gene expression. The goal of this work was to clarify each step of the process of noncytolytic suppression. </p><p>The effector cell was examined to understand the regulation of antiviral factors and to construct a profile of the factors expressed during CNAR. CD8<super>+</super> T-lymphocytes from HIV-1 infected individuals express unidentified factors that suppress viral replication by inhibiting HIV-1 gene expression. Understanding the regulation of these antiviral CD8<super>+</super> T cell-derived factors can provide important insights into how to elicit these factors with therapeutic regimens. For a small subset of human genes, histone deacetylases (HDACs) are epigenetic regulators that condense chromatin to repress transcription. We examined the role of epigenetics in modulating the HIV-1 suppressive factors expressed by primary CD8<super>+</super> T cells from subjects naturally controlling virus replication. HIV-1 suppression by CD8<super>+</super> T-lymphocytes from virus controllers was reversed up to 40% by the addition of an HDAC inhibitor. Therefore, histone deacetylation within CD8<super>+</super> T-lymphocytes was necessary for potent suppression of HIV-1 infection.</p><p>Blocking HDACs impairs the ability of CD8<super>+</super> T-lymphocytes to repress HIV-1 transcription, demonstrating the expression of the suppressive factors is regulated by epigenetics. We used this tool to identify the potential antiviral factors that result in decreased noncytolytic suppression. Through real-time PCR analysis of 164 genes we identified 4 genes in primary CD8<super>+</super> T-lymphocytes from a virus controller, and 12 genes in a CD8<super>+</super> T-cell line that were greatly downregulated in response to a HDAC inhibitor. Additionally, we analyzed the chemokine and cytokine profile of these two cell types to characterize what molecules these cells secrete during CNAR. MIP-1 Beta, MIP-1 Alpha, IP-10, and MIG correlated most strongly with the magnitude of CNAR (<italic>p</italic> < 0.0001). </p><p>The response of the target cell to the antiviral factors was analyzed to better understand how CD8<super>+</super> T cell antiviral factors exert suppressive activity on the HIV-1 genome in an infected cell. Noncytolytic suppression was not dependent on epigenetic changes within the target cells, as HDAC1 within the target cell was dispensable, and histone acetylation at the HIV-1 LTR remained unchanged in the presence of CD8+ T-lymphocytes. </p><p>The genetic elements within HIV-1 and the viral protein Tat were investigated to provide insight into resistance to CNAR. Two virus isolates from the same individual with contrasting sensitivities to CNAR were investigated to identify the genetic elements that confer these phenotypes. Sequence analysis of the two isolates identified mutations in the exon splicing silencers (ESS) 2 and 3 in these viruses. ESS2 and 3 are thought to control splicing of HIV-1 Tat, however levels of spliced Tat RNA levels did not differ between the two isolates. The introduction of the ESS2 mutation into a heterologous HIV-1 isolate moderately boosted resistance to CNAR, suggesting a function for the mutation apart from spliced Tat RNA levels. </p><p>In total, a comprehensive analysis of each component of CNAR is discussed here to enhance the overall understanding of the mechanisms of CNAR.</p> / Dissertation

Chromatin structure is a fundamental determinant of eukaryotic gene expression and it is composed of two chromatin environments, euchromatin and heterochromatin. Euchromatin provides an accessible platform for transcription factors; hence it is permissive for gene expression. Heterochromatin on the other hand is highly compacted and inaccessible, which in most cases leads to transcriptional repression. A locus that is composed of both of these environments is the ribosomal DNA (rDNA). In eukaryotes the rDNA is composed of hundreds to thousands of tandemly repeated genes where maintaining both silent and active copies is fundamental for the stability of the genome. The aim of this research was to investigate the role of the rDNA in gene expression in Drosophila melanogaster. In D. melanogaster the rDNA loci are present on the X and Y chromosomes. This research used the Y-linked rDNA array to investigate the role of this locus on gene expression. A genetic and molecular strategy was designed to create and quantify specific, graded and isogenic Y- linked rDNA deletions. Then the deletions were used to address the effect of rDNA deletions on gene expression using reporter genes sensitive to Position Effect Variegation (PEV). In addition, the effect of the deletions in nucleolus size and structure as well as the effect of spontaneous rDNA deletions on gene expression were tested in this study. This research found that changes in rDNA size change the chromatin balance, which resulted in increased expression of the reporter genes, decreased nucleolus volume, and altered nucleolus structure. These findings prompted a further research question on whether this effect on gene expression occured globally in the genome. This was addressed by performing microarray analysis where the results showed that rDNA deletions affect about half of the genes on the genome. Presented in this dissertation is evidence that suggest a novel role for the rDNA is a global modulator of gene expression and also is a contributor to the gene expression variance observed in natural populations.

Histone methyltransferases are key players in eukaryotic gene regulation. The goal of this thesis was to study the role of the histone methyltransferase Mll2 in developing embryos and adult mice. Targeting of mouse ES cells with a multipurpose allele and blastocyst injection had previously generated a mouse line allowing analysis of Mll2 function by knock-out and conditional mutagenesis using Cre/loxP. The first part of the thesis comprised the analysis of the Mll2-/- phenotype, and included the cloning of a targeting construct to generate an ubiquitous, ligand-regulated Cre line. In the second part, we did conditional mutagenesis using the Rosa26-CreER(T2) line obtained from collaborators, and achieved complete knock-out of Mll2 in most tissues of embryos, neonates and adult mice. Mll2 is essential during embryonic development, as mutant embryos were severely growth retarded, had significant increases in apoptosis, and failed in gestation between E 9.5 and E11. Conditional removal of Mll2 protein at gastrulation (E 6.5) produced a similar phenotype at E 11. In contrast, the absence of Mll2 function after E 11 did not result in obvious defects at E16 and indicates an essential role for Mll2 between E6 and E11. Indeed, we identified a loss of expression of 3 important developmental regulators in mutants of this developmental stage: Hoxb1, Mox1 and Six3 are candidate targets for Mll2 regulation that encode homeobox type transcription factors involved in specifying cellular identity. We observed correct establishment of their developmental expression patterns, which than decay in Mll2-/- mutants at E9.5. These data concord with and extend current thoughts about the fly orthologue of Mll2, Trithorax, which suggest that it acts as an epigenetic lock in chromatin to maintain expression of certain transcription factors key to respective cellular identities, after their expression patterns have been established. After birth, Mll2 is dispensable in most tissues, as conditional knock out in neonates and adult mice did not produce any pathological findings except infertility of mutant males and females. Histological analysis of testis revealed progressive loss of spermatogonia, associated with increases in apoptosis but without overt proliferation, meiotic or differentiation defects or loss of the supporting Sertoli cells. Consequently, in addition to its regulation of homeotic genes during development, Mll2 is required for the maintenance of various mitotic cell populations including ES cells, embryonal cells and germ cells.

Hybrid plants and animals, like corn and the domestic dog, grow larger and more vigorously than their parents, a common phenomenon known as hybrid vigor or heterosis. In hybrids between Arabidopsis ecotypes or species (in allotetraploids), altered expression of circadian clock genes leads to increased starch and chlorophyll content and greater biomass. In plants and animals, circadian clock regulation plays a key role in optimizing metabolic pathways, increasing fitness, and controlling responses to biotic and abiotic stresses. In the allotetraploids, the increased level of heterosis is likely caused by interspecific hybridization as well as genome doubling. However, it is unknown how genome dosage and allelic effects influence heterosis, and whether additional clock output traits, such as stress responses, are altered in hybrids. In three related projects, the effects of genomic hybridization (including parent-of-origin effects) and genome dosage on heterosis were elucidated. In my first project, I found that although ploidy influenced many traits, including seed and cell size, biomass and circadian clock gene expression were most strongly influenced by hybridization. Additionally, parent-of-origin effects between reciprocal hybrids were frequently observed for many traits. In my second project, I described a unique role for RNA-directed DNA methylation (mainly CHH methylation) in mediating the parent-of-origin effect on expression of the circadian clock gene CCA1 in reciprocal hybrids. Altered CCA1 expression peaks were associated with heterosis of biomass accumulation in the reciprocal hybrids. Lastly, I used transcriptome sequencing in hybrids at different times of day to examine changes in downstream clock-regulated pathways. In the hybrids, many genes in photosynthetic pathways were upregulated, while many genes involved in biotic and abiotic stresses were repressed during the morning and afternoon, respectively. Additionally, natural variation between parents in stress-responsive gene expression was found to be crucial for producing vigorous hybrids. These conceptual advances increase the mechanistic understanding of heterosis, and may guide selection of parents for making better hybrids. / text

No description available.

Epigenetic dysfunction is a known contributor in carcinogenesis, and is emerging as a mechanism involved in toxicant-induced malignant transformation for environmental carcinogens such as arsenicals. In addition to aberrant DNA methylation of single genes, another manifestation of epigenetic dysfunction in cancer is agglomerative DNA methylation, which can participate in long-range epigenetic silencing that targets many neighboring genes and has been shown to occur in several types of clinical cancers. Using in vitro model systems of toxicant-induced malignant transformation, we found hundreds of aberrant DNA methylation events that emerge during malignant transformation, some of which occur in an agglomerative fashion. In an arsenite-transformed prostate epithelial cell line, the protocadherin (PCDH), HOXC and HOXD gene family clusters are targeted for agglomerative DNA methylation. Aberrant DNA methylation in general occurred more often within H3K27me3 stem cell domains. We found a striking association between enrichment of H3K9me3 stem cell domains and toxicant-induced agglomerative DNA methylation. Global gene expression profiling of the arsenite-transformed prostate epithelial cells showed that gene expression changes and DNA methylation changes were negatively correlated, but less than 10% of the hypermethylated genes were down-regulated. These studies confirm that a majority of the DNA hypermethylation events occur at transcriptionally repressed, H3K27me3 marked genes. In contrast to aberrant DNA methylation targeting H3K27me3 pre-marked silent genes, we found that actively expressed ZNF genes marked with H3K9me3 on their 3' ends, are preferred targets of DNA methylation linked gene silencing. H3K9me3 mediated gene silencing of ZNF genes was widespread, occurring at individual ZNF genes on multiple chromosomes and across ZNF gene family clusters. At ZNF gene promoters, H3K9me3 and DNA hypermethylation replaced H3K4me3, resulting in a widespread down-regulation of ZNF gene expression which accounted for 8% of all the down-regulated genes in the arsenical-transformed cells. In summary, these studies associate arsenical exposure with agglomerative DNA methylation of gene family clusters and widespread silencing of ZNF genes by DNA hypermethylation-linked H3K9me3 spreading, further implicating epigenetic dysfunction as a driver of arsenical-induced carcinogenesis.

Epigenetics is an important layer of transcriptional control necessary for cell-type specific gene regulation. We developed computational methods to analyze the combinatorial effect and large-scale organizations of genome-wide distributions of epigenetic marks. Throughout this dissertation, we show that regions containing multiple genes with similar epigenetic patterns are found throughout the genome, suggesting the presence of several chromatin domains. In Chapter 1, we develop a hidden Markov model (HMM) for detecting the types and locations of epigenetic domains from multiple histone modifications. We use this method to analyze a published ChIP-seq dataset of five histone modification marks in mouse embryonic stem cells. We successfully detect domains of consistent epigenetic patterns from ChIP-seq data, providing new insights into the role of epigenetics in longrange gene regulation. In Chapter 2, we expand our model to investigate the genome-wide patterns of histone modifications in multiple human cell lines. We find that chromatin states can be used to accurately classify cell differentiation stage, and that three cancer cell lines can be classified as differentiated cells. We also found that genes whose chromatin states change dynamically in accordance with differentiation stage are not randomly distributed across the genome, but tend to be embedded in multi-gene chromatin domains. Moreover, many specialized gene clusters are associated with stably occupied domains. In the last chapter, we develop a more sophisticated, tiered HMM to include a domain structure in our chromatin annotation. We find that a model with three domains and five sub-states per domain best fits our data. Each state has a unique epigenetic pattern, while still staying true to its domain’s specific functional aspects and expression profiles. The majority of the genome (including most introns and intergenic regions) has low epigenetic signals and is assigned to the same domain. Our model outperforms current chromatin state models due to its increased domain coherency and interpretation.