Methyl-CpG Binding Proteins (MCBPs) are thought to function as the interpreters of epigenetic information encoded in cytosine methylation. Their ability to translate DNA methylation into local transcriptional repression has sparked interest in the role of Methyl-Binding Domain Proteins (MBDs) in cancer, where repatterning of CpG methylation is common. In this dissertation I summarize and discuss observations made in the Ginder Lab linking MCBPs to the progression of neoplastic disease. It is clear from our work that the Methyl Binding Domain Protein 2 (MBD2) is necessary for the persistent repression of critical tumor suppressor genes in breast cancer. We show that stable knockdown of MBD2 also leads to growth suppression in cultured human mammary epithelial cancer lines (MCF-7, 49% suppression; MDA-MB-231, 77%; MDA-MB-435, 94%; SK-BR-3, 92%) with the peak cytotoxicity and anti-proliferative effect occurring as late as 2-3 weeks after knockdown. MBD2 knockdown also led to a decrease in viable tumor cells at equivalent doses of the histone deacetylase inhibitor, SAHA (Vorinostat™), and chemotherapeutic agents Doxorubicin, and Paclitaxel. Stable MBD2 knockdown in MCF7 cells led to an increased proportion of normal epithelial structures in 3D culture (70%, [CI=0.55-0.83]) when compared to untransfected (46%, [CI=0.39-0.53], p≤0.038) or scrambled shRNA transfected (37%, [CI=0.29-0.45], p≤0.012) controls. In vivo xenograft studies show tumor growth in BALB/C nu/nu mice was significantly impaired when mice were implanted with human breast cancer cells harboring MBD2 targeted shRNA. Following MBD2 knockdown, tumor suppressor promoter methylation remained unchanged despite sustained increases in gene expression, arguing against the convention that passive demethylation occurs with increased transcription. Our data suggest that uncoupling CpG methylation from histone modifications or other repressor functions by removing MBD2 is sufficient to initiate and maintain anti-tumor gene transcription in the absence of secondary changes in DNA methylation. In this dissertation I present evidence for the pathologic role of MBD2 in breast cancer and provide mechanistic support for the prospect of targeting MBDs in neoplastic disease..

Methyl-CpG-binding proteins (MeCP2, MBD1-MBD3) recruit transcriptional co-repressor molecules to methylated regions and silence transcription. The role of MBD2 in regulating brain function and behavior remains largely unexamined. To begin elucidating whether MBD2 influences neural function, I assessed the behavioral performance of Mbd2 null mice, compared their hippocampal electroencephalographic activity during exploration, and performed protein and mRNA expression assessments. The results indicate that mutant mice display a heightened anxiety-like behavior, diminished explorative activity and reduced sociability compared to wild-type mice. However, these behavioral differences were not paralleled by neurophysiological impairments. Mutant hippocampal and cortical samples display significantly elevated MeCP2 mRNA levels. Yet, MeCP2 protein expression did not mirror the mRNA profile and instead was significantly reduced. Glucocorticoid Receptor mRNA levels were significantly reduced in the hippocampus and cortex regions of Mbd2 null brains. The loss of MBD2 is sufficient to induce behavioral impairments in mice without introducing gross deficits in hippocampal neurophysiology.

Methyl-CpG-binding proteins (MeCP2, MBD1-MBD3) recruit transcriptional co-repressor molecules to methylated regions and silence transcription. The role of MBD2 in regulating brain function and behavior remains largely unexamined. To begin elucidating whether MBD2 influences neural function, I assessed the behavioral performance of Mbd2 null mice, compared their hippocampal electroencephalographic activity during exploration, and performed protein and mRNA expression assessments. The results indicate that mutant mice display a heightened anxiety-like behavior, diminished explorative activity and reduced sociability compared to wild-type mice. However, these behavioral differences were not paralleled by neurophysiological impairments. Mutant hippocampal and cortical samples display significantly elevated MeCP2 mRNA levels. Yet, MeCP2 protein expression did not mirror the mRNA profile and instead was significantly reduced. Glucocorticoid Receptor mRNA levels were significantly reduced in the hippocampus and cortex regions of Mbd2 null brains. The loss of MBD2 is sufficient to induce behavioral impairments in mice without introducing gross deficits in hippocampal neurophysiology.

De nombreux gènes suppresseurs de tumeurs sont inactivés par hyperméthylation dans les cancers. Cette inactivation serait en partie initiée par la protéine, MBD2 (Methyl-CpG-Binding Domain protein 2). Cette protéine recrute au niveau de séquences méthylées des complexes enzymatiques capables de modifier la structure chromatinienne et crée ainsi des régions fonctionnellement inactives. Dès lors, ce répresseur apparaît être une cible potentielle pour combattre le cancer. Dans cette perspective, rechercher les cibles de MBD2 et comprendre sa capacité à contrôler l’expression génique semblent cruciales. Au cours de deux études gènes candidats, nous avons pu démontrer (i) une réelle spécificité de cible du répresseur méthylationdépendant MBD2 pour les loci hTERT et pS2/TFF1 ; et (ii) un nouveau rôle de la protéine MBD2 en tant que modulateur de l’expression génique. De plus, les actions antagonistes entre le répresseur MBD2 et le trans-activateur naturel du gène pS2, le récepteur aux oestrogènes α, ont été explorées. Puis, l’analyse globale des profils de distribution de MBD2, de la méthylation de l’ADN, ainsi que de l’ARN polymérase II, sur puce promoteur a montré que MBD2 possède toutes les caractéristiques d’un répresseur trancriptionnel méthylation-dépendant. En effet, 74% des promoteurs fixés par MBD2 sont méthylés et cette liaison est associée dans 65% des cas à une répression transcriptionnelle. / In the past few years, several clinical trials have shown that targeting DNA methylation machinery might be of interest in cancer therapy to restore tumor suppressor genes expression and inhibit tumor growth. The Methyl-CpG-Binding Domain protein 2 (MBD2) is an important constituent of the DNA methylation machinery since this protein is directly involved in the mediation of the epigenetic signal. Moreover, MBD2 seems to show some gene specificity, its inhibition reactivate a limited number of genes. Taken together these data suggest that MBD2 represents potential new target in cancer therapy and, therefore, new insights on MBD2 specificities are, in this context, of importance. To this end, we have developed two different approaches: a candidate genes analysis and a genome-wide analysis, using ChIP-on-chip method, in order to map MBD2 binding sites. The candidate gene approaches are strongly in favour of the “one gene – one MBD” hypothesis, at least for the genes analyzed. Indeed, our results indicate that MBD2 is specifically and directly involved in the transcriptional repression of hTERT and pS2/TFF1 genes. Furthermore, a new role of MBD2 in the fine-scale modulation of these genes was demonstrated, and the antagonist actions between MBD2 and the natural trans-activator of pS2 gene, the estrogen α, were explored. Genome wide distribution of MBD2 binding sites, DNA Methylation profiles, and silencing potential, showed that the MBD2 is a real methylation-dependant transcriptional repressor: 74% of the MBD2 binding promoters are methylated and 65% silenced.

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.

The reexpression of the fetal γ-globin gene in adult erythrocytes is of therapeutic interest due to its ameliorating effects in β-hemoglobinopathies. We recently showed that Methyl CpG Binding Domain Protein2 (MBD2) contributes to the silencing of the chicken embryonic ρ-globin and human fetal γ-globin genes. We further biochemically characterized an erythroid MeCP1 complex that is recruited by MBD2 to mediate the silencing of these genes. These observations suggest that the disruption of the MeCP1 complex could augment the expression of the fetal/embryonic globin genes. In the studies presented in chapter 2, we have pursued a structural and biophysical analysis of the interaction between two of the six components of the MeCP1 complex: MBD2 and p66α. These studies show that the coiled coil regions from MBD2 and p66α form a highly stable heterodimeric complex. Further, overexpressing the p66α coiled coil domain in adult erythroid cells can augment the expression of the chicken ρ-globin and human γ-globin genes, by disrupting the assembly of a functional MeCP1 complex. This indicates that the exogenously expressed p66α coiled coil peptide competes with the endogenous p66α for the interaction with the coiled coil domain of MBD2. These studies show that the coiled coil interaction between MBD2 and p66α could serve as a potential targets for the therapeutic induction of fetal hemoglobin. The laboratory showed that knockout of MBD2 in transgenic mice carrying the human β-globin gene cluster, results in an elevated expression of γ-globin in adult erythrocytes. However, MBD2 does not directly bind to the γ-globin gene to mediate its silencing. In the work presented in chapter 3, we have tested the hypothesis that MBD2 may suppress γ-globin gene transcription in adult erythrocytes indirectly, by binding to and repressing transcription of intermediary gene/s which may be involved in γ-globin gene regulation. Employing microarray technology, we have identified Gab1 and ZBTB32 as candidate genes that may be involved in the MBD2 mediated silencing of γ-globin.

The genes of the human β-globin locus are located on chromosome 11 in the order of their expression during development: 5' ε, γ, β 3'. During development, silencing of the 5' gene occurs with activation of the immediate 3' gene. This process occurs twice and is termed hemoglobin switching. The exact mechanism(s) of this process have not been fully described. Herein, we describe a role for DNA methylation and methyl binding domain protein 2 in the transcriptional regulation of the human embryonic and fetal beta type globin genes. Adult mice containing the entire human β-globin locus as a yeast artificial chromosome (βYAC) express very low levels of the fetal γ-globin gene. However, treatment of adult βYAC transgenic mice with the DNA methyltransferase inhibitor, 5-azacytidine, induces a >10-fold increase γ-globin mRNA levels. In addition, βYAC transgenic mice null for methyl binding domain protein 2 (MBD2) express a similar level of γ-globin mRNA. DNA methylation and MBD2 appear to induce γ-globin expression via the same pathway(s), as treatment of MBD2 null βYAC transgenic mice do not show an additive boost in γ-globin expression. MBD2 does not bind to the γ-globin promoter region in vivo indicating MBD2 mediated transcriptional silencing does not occur by recruitment of transcriptional repression complexes to the γ-globin gene promoter. Additionally, these transgenic mice contain only the 5' portion of the β-globin locus through the ε-globin, and do not express the ε-globin genes as adults. However, treatment with 5-azacytidine or loss of MBD2 induces expression of the ε-globin gene in adult transgenic mice. A similar induction of ε-globin is seen in βYAC transgenic mice under the same conditions. The level of expression of the ε-globin gene is much lower than the γ-globin gene, indicating the powerful effect of the cis elements mediating transcriptional repression of the ε-globin gene. These studies indicate DNA methylation and MBD2 contribute to the transcriptional repression of the human embryonic and fetal β-type globin genes. Additionally, MBD2 has been identified as a potential target for the therapeutic induction of fetal hemoglobin for the treatment of hemoglobinopathies.

De nombreux gènes suppresseurs de tumeurs sont inactivés par hyperméthylation dans les cancers. Cette inactivation serait en partie initiée par la protéine, MBD2 (Methyl-CpG-Binding Domain protein 2). Cette protéine recrute au niveau de séquences méthylées des complexes enzymatiques capables de modifier la structure chromatinienne et crée ainsi des régions fonctionnellement inactives. Dès lors, ce répresseur apparaît être une cible potentielle pour combattre le cancer. Dans cette perspective, rechercher les cibles de MBD2 et comprendre sa capacité à contrôler l'expression génique semblent cruciales. Au cours de deux études gènes candidats, nous avons pu démontrer (i) une réelle spécificité de cible du répresseur méthylationdépendant MBD2 pour les loci hTERT et pS2/TFF1 ; et (ii) un nouveau rôle de la protéine MBD2 en tant que modulateur de l'expression génique. De plus, les actions antagonistes entre le répresseur MBD2 et le trans-activateur naturel du gène pS2, le récepteur aux oestrogènes α, ont été explorées. Puis, l'analyse globale des profils de distribution de MBD2, de la méthylation de l'ADN, ainsi que de l'ARN polymérase II, sur puce promoteur a montré que MBD2 possède toutes les caractéristiques d'un répresseur trancriptionnel méthylation-dépendant. En effet, 74% des promoteurs fixés par MBD2 sont méthylés et cette liaison est associée dans 65% des cas à une répression transcriptionnelle.

The human GI tract has evolved to simultaneously absorb nutrients and be the frontline in host defence. These seemingly mutually exclusive goals are achieved by a single cell thick epithelial barrier, and a complex resident immune system which lives in symbiosis with the intestinal microflora and is also able to rapidly respond to invading pathogens. An immunological balance is therefore required to permit tolerance to the normal intestinal microflora, but also prevent the dissemination of pathogenic micro-organisms to the rest of the host. Inappropriate immune responses in genetically susceptible individuals are the hallmark of human inflammatory bowel disease (IBD) and are thus targeting effector immune cells and their cytokines remains the mainstay of treatment. However despite vigorous efforts to delineate the genetic contribution to IBD disease susceptibility using large multinational cohorts, the majority of disease heritability remains unknown. Epigenetics describes heritable changes in chromatin that are not conferred by DNA sequence. These incorporate changes to histones, chromatin structure and DNA methylation, which confer changes to gene transcription and thus gene expression and cellular function. Methylbinding proteins (MBD) have the ability to bind to methylated DNA and recruit large chromatin remodeling complexes that underpin a variety of epigenetic modifications. Methyl- CpG-binding domain protein 2 (MBD2) is one such MBD that is required for appropriate innate (dendritic cell) and adaptive (T cell) immune function, though its role has not been investigated in the GI tract. We hypothesized that alterations in chromatin are central to the reprogramming of normal gene expression that occurs in disease states. By defining the phenotype of immune cells in the absence of MBDs we hope to understand the mechanisms of chromatin-dysregulation that lead to immune-mediated diseases such as IBD. We therefore aimed to assess the role of MBD2 in colon immune cells in the steady state and in murine models of GI tract inflammation, thereafter identifying the culprit cell types and genes responsible for any observed changes. We envisaged that investigating heritable, epigenetic changes in gene expression that are inherently more amenable to environmental manipulation than our DNA code, may provide novel insight to a poorly understood mechanism of disease predisposition. In addition identifying the cellular and gene targets of Mbd2 mediated changes to immune homeostasis that may provide exciting and novel approaches to therapeutic modulation of pathological inflammatory responses. In chapter 3 we assessed the expression of Mbd2/MBD2 in the murine/human GI tract. Consistent with existing mouse data, levels of Mbd2 mRNA increased between anatomical divisions of small (duodenum, ileum, terminal ileum) and large intestine (caecum, colon, rectum). In addition MBD2 mRNA was greater in the rectum versus ileum, with active IBD associated with lower rectal MBD2 mRNA compared to quiescent IBD controls. Thus we sought to understand the role of Mbd2 in the colon, where mRNA levels were the highest in the GI tract and where appropriate immune function is central to prevent damaging inflammation. To address these aims required the development of existing methods of cell surface marker expression analysis using flow cytometry techniques to simultaneously identify multiple innate and adaptive immune populations. Using naïve Mbd2 deficient mice (Mbd2-/-) we observed CD11b+ CD103+ DCs were significantly reduced in number in Mbd2 deficiency. To understand the role of Mbd2 in colonic inflammation we employed a mouse model of chemical (DSS) and infectious (T. gondii) colitis comparing Mbd2-/- and littermate controls (WT). Mbd2-/- were extremely sensitive to DSS and T. gondii mediated colonic inflammation, characterized by increased symptom score, weight loss and histological score of tissue inflammation (DSS) and increased antibody specific cytokine responses (T. gondii) in Mbd2 deficient animals. Flow cytometry analysis of colon LP cells in both infectious and chemical colitis revealed significant accumulation of monocytes and neutrophils in Mbd2-/-. Indeed monocytes and neutrophils were the principal myeloid sources of IL-1b and TNF in DSS colitis and the number of IL-1b/TNF+ monocytes/neutrophils was significantly greater in Mbd2-/-. Lastly we employed our colon LP isolation techniques to analyse immune populations in active and quiescent IBD and healthy controls, using endoscopically acquired biopsy samples. Analysis revealed that as in murine colitis, active human IBD is characterized by the accumulation of CD14High monocyte-like cells, with an associated increased ratio of macrophage:monocyte-like cells. In Chapter 4 we sought to understand the cellular sources of Mbd2 that may explain the predisposition of Mbd2-/- to colitis. Firstly we restricted Mbd2 deficiency to haematopoietic cells using grafting Mbd2-/- bone marrow (BM) into lethally irradiated WT mice. These animals treated with DSS displayed increased weight loss, symptom score, neutrophil accumulation and histopathology score compared to mice irradiated and grafted with WT BM. Given the accumulation of monocytes in Mbd2-/- DSS treated mice, and existing literature supporting a pathogenic role in this model, we then investigated the role of Mbd2 in monocyte function. Colon monocytes sorted from Mbd2-/- and WT DSS treated mice displayed similar expression for many pro-inflammatory genes (Il6, Il1a, Il1b, Tnf), but demonstrated significantly dysregulated expression for some others (Regb, Lyz1, Ido1, C4a). To investigate this in a more refined model, we lethally irradiated WT mice and repopulated them with a WT:Mbd2-/- BM mix. This enabled the analysis of WT and Mbd2-/- haematopoietic cells in the same animal. Colon WT and Mbd2-/- monocyte recruitment and cytokine production in DSS treated mixed BM chimeras was equivalent between genotypes suggesting that Mbd2 deficiency in monocytes alone did not explain the increased susceptibility of Mbd2-/- to DSS colitis. We then restricted Mbd2 deficiency to CD11c expressing cells, given the known role for Mbd2 in their function, and for CD11c+ cells in DSS, using a CD11cCreMbd2Fl/Fl system. DSS treated mice with Mbd2 deficient CD11c+ cells demonstrated increased weight loss, symptoms score, histolopathology score, monocyte and neutrophil colon accumulation compared to controls. To further explore the role of Mbd2 in colon CD11c+ cells, macrophage and DCs from DSS treated WT and Mbd2-/- mice were purified and their gene expression analysed. Mbd2-/- versus WT macrophages demonstrated significantly altered expression of both pro- (Il1a, C6, Ido1, Trem2) and antiinflammatory (Tgfbi, Retnla) pathways that we hypothesized was a method for attempted host control of excessive colon damage in Mbd2-/- mice. DC gene expression analysis was hampered by small sample size, but demonstrated a large number of small expression changes, including IL-12/IL-23 (Jak2) and autophagy (Lrrk2) pathways. Lastly levels of costimualtory molecules (CD40/CD80) were increased in Mbd2-/- but not CD11cΔMbd2 colon LP DCs/macrophages suggesting that non-CD11c+ cellular sources of Mbd2 were required to produce increased activation phenotype in these cells. Finally in Chapter 5 we explored the role for Mbd2 in non-haematopoietic cells, namely the colonic epithelium. Here we first developed a novel method for identifying and purifying these cells using flow cytometry. Mbd2 deficient colonic epithelium demonstrated increased expression of activation markers MHC II and LY6A/E in the steady state and in DSS / T. muris mediated colonic inflammation. Indeed FACS purified colon epithelial cells from naive and DSS treated, Mbd2-/- and WT mice revealed conserved dysregulated gene expression independent of inflammation: Both naïve and inflamed Mbd2 deficient epithelium displayed significantly increased expression of genes responsible for antigen processing/presentation (MHC I, MHC II, immunoproteasome) and decreased expression of genes involved in cell-cell adhesion (Cldn1, Cldn4). Lastly we investigated whether the observed differences in Mbd2-/- cell types conferred alterations in the makeup of the intestinal microflora. Interestingly independent of co-housing of Mbd2-/- and WT animals, Mbd2 deficiency consistently predicted the microbial composition, with increased levels of Clostridales and decreased levels of Parabacteroides bacteria. Collectively we have identified CD11c+ cells, monocytes and colon epithelial cells as key cell types for Mbd2 mediated changes in gene expression that affect mucosal immune responses. These data thus identify Mbd2 gene targets within these cell types as exciting new areas for investigation and therapeutic modulation to limit damaging GI tract inflammation.

La méthylation de l'ADN est une marque épigénétique répressive impliquée dans de nombreux processus physiologiques et pathologiques. Des hyperméthylations de promoteurs sont ainsi responsables de répressions transcriptionnelles de gènes suppresseurs de tumeurs dans les cancers. La méthylation de l'ADN serait capable d'induire une répression transcriptionnelle par la combinaison de deux mécanismes principaux : l'éloignement de facteurs de transcription activateurs, et le recrutement de protéines répressives liant spécifiquement l'ADN méthylé. MBD2 est une protéine de liaison à l'ADN méthylé capable de recruter les complexes répresseurs NuRD et SIN3A. ZBTB4 est capable de se lier à l'ADN méthylé in vitro et induit une répression de la transcription de plasmides méthylés lorsqu'elle est surexprimée. Son rôle de répresseur transcriptionnel dépendant de la méthylation de l'ADN reste toutefois peu documenté. Nous avons identifiés par RNAseq les modifications du transcriptome induites par une déplétion de MBD2 ou de ZBTB4. Les gènes surexprimés après déplétion de MBD2 ou ZBTB4 sont méthylés sur leur promoteur, et sont aussi surexprimés après traitement avec des agents déméthylants. Des résultats d'immuno-précipitations de chromatine réalisées contre les deux protéines endogènes montrent que la quasi-totalité des sites de fixation de MBD2 et qu'une partie des sites de fixations de ZBTB4 correspondent à des régions méthylés. Ces résultats confirment à l'échelle du génome que MBD2 endogène est bien un interprète majeur de la méthylation de l'ADN, et que ZBTB4 réprime bien la transcription de gènes méthylés. Nous avons aussi observé une redistribution importante de MBD2 sur le génome dans des modèles de progression tumorale. Nos résultats montrent que les gènes réprimés pendant la transformation oncogénique le sont en partie par MBD2. L'expression de certains de ces gènes peut être induite dans les lignées transformées par déplétion de MBD2 par siRNA / DNA methylation is an epigenetic mark that plays a role in many physiological and pathological processes. Indeed, silencing of tumor suppressor genes in cancer is frequently caused by promoter hypermethylations. Transcriptional repression induced by DNA methylation is likely caused by the combination of two mechanisms: the repulsion of activator transcription factors, and the recruitment of repressor proteins able to specifically recognize methylated DNA. MBD2 is a methyl DNA binding protein that cans recruits NuRD or SIN3A repressor complexes. ZBTB4 is able to bind methylated DNA in vitro, and can repress the transcription of methylated plasmids when overexpressed. Its methylationdependent transcriptional repressor function remains poorly documented. By RNAseq, we have identified transcriptomic modifications induced by the depletion of either MBD2 or ZBTB4. Genes up regulated after MBD2 or ZBTB4 depletion were methylated on their promoter, and were also up regulated after treatment with demethylating agents. Chromatin immunoprecipitations experiments against endogenous proteins showed that almost all MBD2 binding sites, and that a part of ZBTB4 binding sites, correspond to methylated DNA regions. These results confirmed at genome wide scale that endogenous MBD2 is a major reader of DNA methylation and that ZBTB4 does repress the transcription of methylated genes. We observed an important redistribution of MBD2 on the genome in models of tumor progression. Our results showed that MBD2 plays role in gene repressions occurring during oncogenic transformation. Some of those repressed genes can be re-expressed in transformed cell lines after depletion of MBD2 by siRNA