Methyl-CpG binding protein 2 (MeCP2) was discovered as a protein binding to methylated DNA more than 20 years ago. It is very abundant in the brain and was shown to be able to repress transcription. The mutations in MeCP2 cause Rett syndrome, an autism-spectrum neurological disorder affecting girls. Yet, the exact role of MeCP2 in Rett disease, its function and mechanism of action are not fully elucidated. In order to shed some light on its role in the disease the aim of this project was to identify proteins interacting with MeCP2. Affinity purification of MeCP2 from mouse brains and mass spectrometry analysis revealed new interactions between MeCP2 and protein complexes. Detailed analysis confirmed the findings and narrowed down the top interactions to distinct regions of MeCP2. One of the domains interacts with identified NCoR/SMRT co-repressor complex and is mutated in many patients with Rett syndrome. In vitro assays proved that these mutations abolish the putative transcriptional repressor function of MeCP2. We propose a model in which Rett syndrome is caused by two types of mutations: either disrupting the interaction with DNA or affecting the interaction with the identified complex, which has an effect on the global state of chromatin. The presented findings can help to develop new therapies for Rett syndrome in the future.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

The phenomenon of cell type-specific DNA methylation has received much attention in recent years and a number of DNA methylation differences have been described between cells of the immune system. Of particular interest when studying DNA methylation are CpG islands (CGIs) which are distinct from the rest of the genome due to their elevated CpG content, generally unmethylated state and promoter association. In the instances when they become methylated this is associated with gene repression although it is unclear the extent to which differential methylation corresponds to differential gene expression. I have used an immune system model to assess the role of CGI methylation and the role of the methylation reader MBD2 in regulation of gene expression. A relatively small number of DNA methylation differences were seen between immune cell types with the most developmentally related cells showing the fewest methylation differences. Interestingly, the vast majority of CGI-associated cellspecific methylation occurred at intragenic CGIs located, not at transcription start sites, but in the gene body. Increased intragenic CGI methylation tended to associate with gene repression, although the precise reason for this remains unclear. Most differentially methylated CGIs were depleted for the active chromatin mark H3K4me3 regardless of their methylation state but some of these were associated with the silencing mark H3K27me3 when unmethylated. These findings suggest that intragenic CGIs are a distinct class of genomic element particularly susceptible to cell type-specific methylation. I also looked at the effect of removing the methyl- CpG binding domain protein MBD2 from immune system cells. Immune cells from Mbd2-/- mice showed a number of previously uncharacterised phenotypes as well as a number of differences in gene expression compared to wild-type animals. Most of these genes increased their expression in the absence of MBD2 consistent with MBD2’s role as a transcriptional repressor and Mbd2-/- Th1 cells showed increases in histone H3 acetylation compared to wild-type Th1 cells. This work provides an insight into the role played by cell-specific CGI methylation and MBD2 in regulating gene expression.

In higher eukaryotes, the DNA base Cytosine can exist in a variety of modified forms when in the dinucleotide CpG. Although a methylated form tends to dominate within the genome, approximately 1% of all CpG dinucleotides are found unmodified at high densities spanning around 1Kb and tend to co-localise to the 5’ ends of around 60% of annotated gene promoters. These unique DNA sequences are known as CpG islands (CGIs) and their role within the genome to date is largely unknown. Methylation of CGIs in cancers however has been linked to silencing of associated genes implying a role in gene regulation. Furthermore these sites are also interesting as they remain specifically nonmodified within a genome rich in methylated CpG. We set out to better understand the roles for CGIs through the characterisation of any specific CGI binding proteins. Digestion of nuclei with methyl sensitive restriction enzymes facilitates the purification of CGI fragments. Subsequent immunohistochemistry on the CGI chromatin fragments along with ChIP-PCR over several CGIs revealed an enrichment of the “active” histone modifications including H3K4me3, a depletion of the “silencing” marks such as H3K27me3, as well as a group of CGI specific binding factors. These latter proteins contained a domain previously shown to bind to non-methylated CpG dinucleotides (the CXXC domain) and as such were ideal candidates for CGI specific factors, in particular a protein called Cfp1. Genome wide sequencing revealed a striking correlation between Cfp1 and H3K4me3 which were both seen at around 80% of islands. Furthermore, the presence of Cfp1/H3K4me3 at islands tended to have a negative correlation with the presence of chromatin rich in the silencing histone modification H3K27me3. Closer investigation of the Cfp1 protein reveals it to be a true non-methyl CGI binding factor in vivo and shRNA reduction of Cfp1 levels to around 10% of wild type resulted in a precipitous drop in H3K4me3 levels over CGIs without a dramatic reduction in global H3K4me3 levels. As Cfp1 has been shown to be part of the Set1 histone H3K4 methyltransferase complex responsible for this modification, this CXXC protein may be attracting this histone modifying complex and as such represents a method whereby the underlying DNA sequence (CpG) can drive the overlying epigenetic state. This study may go some way to understanding the functional significance of CGIs within the genome.

Prostate cancer is the most common non-skin cancer in America, and the most commonly diagnosed cancer among males. When metastatic, the disease can ultimately be incurable. Consequently, alternative strategies to current treatments are sought, especially in the area of immunotherapy. Vaccine immunotherapy using a specific antigen, such as prostate specific antigen (PSA) seeks to stimulate both the innate and adaptive immune system to destroy tumor cells in the body. PSA is an ideal target antigen given that it has a narrow distribution in tissues and is expressed in virtually all prostate cancer cases. An adenovirus encoding for PSA (Ad-PSA) can be used to deliver the genomic data encoding for PSA production and secretion to the target cell. This type of viral gene delivery system has already been shown to have the potential to stimulate anti-tumor activity. To enhance this activity and increase transfection efficiency, we proposed the combination of a viral system with a non-viral system, in the form of a cationic polymer such as poly(ethyl)enimine (PEI) or chitosan. Cationic polymers complex with the negatively charged adenovirus to form nanoparticles that can be used in gene delivery. Delivery in nanoparticle form can give enhanced uptake by the antigen-presenting cells necessary to initiate the targeted immune response. To further augment this response, previous research has shown that CpG sequences act as an adjuvant to enhance the efficacy of the Ad-PSA vaccines' tumor protection. CpG delivered in particulate form has also been shown to be more effective than delivery in solution. The objective of this proposal was to test the hypothesis that co-delivery of this targeted viral/non-viral gene delivery system will enhance tumor protection in a mouse model of prostate cancer. Using the OVA model antigen system, we found that the adenovirus encoding OVA (AdOVA), coupled with the polymer PEI, enhanced tumor protection in vivo compared to AdOVA alone. To move towards our therapeutic model, these experiments were repeated using chitosan as the cationic polymer carrier, delivering AdOVA, and incorporating CpG into some particles. In this set of experiments, we found that AdOVA + CpG gave the best tumor protection in challenge studies. AdOVA + chitosan + CpG showed a decrease in protective levels and numbers of antigen-specific immune cells. Further experiments focused on elucidating the mechanisms by which chitosan and CpG modulate the immune response. Using the therapeutic AdPSA model, chitosan was not found to enhance tumor protection or numbers of antigen-specific immune cells. Additional experiments found that this depression was not due to problems with viral infectivity or secretion due to chitosan complexation. A series of kinetics studies were performed which showed that peak levels of effector T cells were present 14 days later in AdPSA + CpG immunized mice than in AdPSA alone. This delayed effect may explain the increased levels of protection in AdPSA + CpG mice, and be useful in future vaccine design concerning the timing of peak response.

As respiratory syncytial virus (RSV) is a respiratory pathogen that causes significant morbidity and mortality in infants, there has always been great interest in the development of a vaccine. In the 1960s, children were immunized with formalin-inactivated (FI)-RSV vaccines. Not only did these vaccines fail to prevent infection, but in most cases they resulted in enhanced disease upon subsequent exposure to the virus. In the intervening years, studies in mice have led to the hypothesis that the enhanced disease is due to an aberrant Th2-biased immune response. Thus, we hypothesized that formulating FI-RSV vaccines with a Th1 promoting adjuvant, such as CpG oligoeoxynucleotides (ODN), would result in the induction of protective immunity against RSV without risk of deleterious effects. We observed in calves that parenterally delivered FI-bovine RSV (BRSV) formulated with CpG ODN resulted in a shift towards a Th1-biased or more balanced immune response that was protective against BRSV.<p> As RSV infects the lung mucosa, vaccines that induce mucosal immunity are desirable. Parenterally delivered vaccines typically induce systemic immunity with low mucosal immune response levels, whereas mucosally delivered vaccines induce systemic and mucosal immunity. However, upon mucosal delivery there is an increased chance of vaccine components being degraded or washed away prior to the induction of immunity. Thus, we added polyphosphazenes (PP) to our mucosal vaccine formulations. PP are synthetic polymers that form non-covalent complexes with other vaccine components, increasing their stability. Intranasally delivered FI-BRSV co-formulated with CpG ODN and PP performed better than FI-BRSV alone, or FI-BRSV formulated with either adjuvant individually, in terms of inducing protective immunity against BRSV in mice. Furthermore, mice that received intranasally-delivered FI-BRSV or BRSV F protein co-formulated with CpG ODN and PP developed higher levels of immunity and protection than mice that received parenterally delivered vaccines. Because of the similarities between BRSV and HRSV, co-formulation of intranasally delivered HRSV vaccines with CpG ODN and PP could prove important in the development of a safe vaccine against HRSV in humans.

A new paradigm for the treatment of infectious disease is through the modulation of innate immune responses. In this capacity, host defense peptides (HDPs) and synthetic Toll-like receptor 9 (TLR9) ligands have the greatest demonstrated potentials. The work presented here considers mechanisms for the improvement of these treatments through optimization, or in the case of HDPs the minimization, of the interactions of these ligands with sensory receptors.<p>Toll-like Receptor 9 activates the innate immune system in response to microbial DNA or immune-modulating oligodeoxynucleotides. While cell stimulation experiments demonstrate the preferential activating ability of CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions regarding the sequence-specificity of TLR9 ligand binding. To address this discrepancy the characterization of human TLR9 ligand binding properties is reported. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity of the receptor is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. <p>Host defense peptides are among the leading candidates to combat antibiotic resistant bacterial strains. Recently, HDPs have been demonstrated to function as ligands for the bacterial sensory kinase PhoQ resulting in the induction of virulence and adaptive responses. Thus, concerns have been raised regarding therapeutic applications of HDPs. Here a methodology is described that permits discrimination and quantification of the distinct, but related, peptide behaviors of direct antimicrobial activity and PhoQ ligand potential. Utilizing peptide derivatives of the model HDP Bac2A it is demonstrated that antimicrobial efficiency is significantly, and inversely, related to PhoQ ligand efficacy. This provides a rational basis for HDP selection with greater therapeutic potential and minimized potential for initiation of bacterial resistance.

A new paradigm for the treatment of infectious disease is through the modulation of innate immune responses. In this capacity, host defense peptides (HDPs) and synthetic Toll-like receptor 9 (TLR9) ligands have the greatest demonstrated potentials. The work presented here considers mechanisms for the improvement of these treatments through optimization, or in the case of HDPs the minimization, of the interactions of these ligands with sensory receptors.<p>Toll-like Receptor 9 activates the innate immune system in response to microbial DNA or immune-modulating oligodeoxynucleotides. While cell stimulation experiments demonstrate the preferential activating ability of CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions regarding the sequence-specificity of TLR9 ligand binding. To address this discrepancy the characterization of human TLR9 ligand binding properties is reported. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity of the receptor is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. <p>Host defense peptides are among the leading candidates to combat antibiotic resistant bacterial strains. Recently, HDPs have been demonstrated to function as ligands for the bacterial sensory kinase PhoQ resulting in the induction of virulence and adaptive responses. Thus, concerns have been raised regarding therapeutic applications of HDPs. Here a methodology is described that permits discrimination and quantification of the distinct, but related, peptide behaviors of direct antimicrobial activity and PhoQ ligand potential. Utilizing peptide derivatives of the model HDP Bac2A it is demonstrated that antimicrobial efficiency is significantly, and inversely, related to PhoQ ligand efficacy. This provides a rational basis for HDP selection with greater therapeutic potential and minimized potential for initiation of bacterial resistance.

As respiratory syncytial virus (RSV) is a respiratory pathogen that causes significant morbidity and mortality in infants, there has always been great interest in the development of a vaccine. In the 1960s, children were immunized with formalin-inactivated (FI)-RSV vaccines. Not only did these vaccines fail to prevent infection, but in most cases they resulted in enhanced disease upon subsequent exposure to the virus. In the intervening years, studies in mice have led to the hypothesis that the enhanced disease is due to an aberrant Th2-biased immune response. Thus, we hypothesized that formulating FI-RSV vaccines with a Th1 promoting adjuvant, such as CpG oligoeoxynucleotides (ODN), would result in the induction of protective immunity against RSV without risk of deleterious effects. We observed in calves that parenterally delivered FI-bovine RSV (BRSV) formulated with CpG ODN resulted in a shift towards a Th1-biased or more balanced immune response that was protective against BRSV.<p> As RSV infects the lung mucosa, vaccines that induce mucosal immunity are desirable. Parenterally delivered vaccines typically induce systemic immunity with low mucosal immune response levels, whereas mucosally delivered vaccines induce systemic and mucosal immunity. However, upon mucosal delivery there is an increased chance of vaccine components being degraded or washed away prior to the induction of immunity. Thus, we added polyphosphazenes (PP) to our mucosal vaccine formulations. PP are synthetic polymers that form non-covalent complexes with other vaccine components, increasing their stability. Intranasally delivered FI-BRSV co-formulated with CpG ODN and PP performed better than FI-BRSV alone, or FI-BRSV formulated with either adjuvant individually, in terms of inducing protective immunity against BRSV in mice. Furthermore, mice that received intranasally-delivered FI-BRSV or BRSV F protein co-formulated with CpG ODN and PP developed higher levels of immunity and protection than mice that received parenterally delivered vaccines. Because of the similarities between BRSV and HRSV, co-formulation of intranasally delivered HRSV vaccines with CpG ODN and PP could prove important in the development of a safe vaccine against HRSV in humans.

The objective of this investigation was to evaluate the effects of administering CpG-ODN to commercial strain chickens as a potential adjuvant to vaccination against Salmonella, Eimeria spp., and Newcastle disease virus, or immunization to bovine serum albumin (BSA). During Experiment 1, which evaluated the dual application of CpG-ODN and a Newcastle disease virus vaccine, in the first of three replicate trials, on day 28 of the experiment, animals in the Vaccine + CpG 1& 14 experimental group were observed to have the highest levels of (p<0.05) anti-NDV IgG in serum. These levels were elevated above levels in animals from all other experimental groups. This suggestion for an adjuvant effect associated with CpG-ODN administration was not supported in the remaining two trials of experiment 1. Experiment 2 evaluated the potential for CpG-ODN to adjuvant a commercial live oocyst coccidial vaccine when applied by an oral route to neonatal broiler chickens. Overall, when body weight gain during challenge, development of intestinal lesions, and anti-Eimeria IgG levels were evaluated, vaccine administration alone was demonstrated to provide the best measure of protection among animals in all experimental groups, including those receiving either CpG-ODN or Non CpG-ODN. Experiment 3 investigated the simultaneous administration of CpG-ODN or Non-CpG ODN and a commercially acquired Salmonella typhimurium vaccine to SCWL chickens. Similar to experiments 1 and 2, antigen specific IgG responses in serum and indices of protection against field strain Salmonella challenge were variable and inconsistent. Anti-BSA IgG levels were compared in broiler and SCWL chickens immunized against BSA by a drinking water route of administration alone, or in combination with two different concentrations of CpG-ODN or Non CpG-ODN in experiment 4. The only observation where CpG-ODN and BSA co-administration resulted in anti-BSA IgG levels that were elevated above BSA alone immunized chickens was measured in broilers at day 19 post-final immunization. Taken together, given the variable results reported in this investigation related to the co-administration of ODN and vaccine or protein antigen, these data are largely inconclusive for suggesting that CpG-ODN can effectively adjuvant humoral immune responses in commercial strain chickens.