DNA methylation-dependent gene silencing, catalyzed by DNA methyltransferases (DNMTs) and mediated by methyl binding domain proteins (MBDs) and histone deacetylases (HDACs), is essential for mammalian development, with the nervous system demonstrating particular sensitivity to perturbations. Little is known, however, about the role of DNA methylation in the stage-specific differentiation of neurons. In the olfactory epithelium (OE), where neurogenesis is continuous and the cells demonstrate a laminar organization with a developmental hierarchy, we identified sequential, transitional stages of differentiation likely mediated by different DNMT, MBD and HDAC family members. Biochemically, HDAC1 and HDAC2 associate with repressor complexes recruited by both MBD2 and MeCP2. HDAC1 and HDAC2, however, are divergently expressed in the OE, a pattern that is recapitulated in the brain. Rather than simultaneous inclusion in a complex, therefore, the individual association of HDAC1 or HDAC2 may provide specificity to a repressor complex in different cell types. Furthermore, distinct transitional stages of differentiation are perturbed in the absence of MBD2 or MeCP2. MeCP2 is expressed in the most apical immature olfactory receptor neurons (ORNs), and is up-regulated with neuronal maturation. In the MeCP2 null OE there is a transient delay in ORN maturation and an increase in neurons of an intermediate developmental stage. Two protein variants of MBD2 are expressed in the OE, with MBD2b expressed in cycling progenitor cells and MBD2a in the maturing ORNs. MBD2 null ORNs undergo increased apoptotic cell death. There is also a significant increase in proliferating progenitors in the MBD2 null OE, likely due, at least in part, to feedback from the dying ORNs, acting to up-regulate neurogenesis. Increased cell cycling in the MBD2 null is also observed post-lesion, however, in the absence of feedback back from the ORNs, a phenotype that is recapitulated by an acute inhibition of HDACs with valproic acid. Therefore, disruptions at both transitional stages of ORN differentiation are likely in the MBD2 null mouse. Together, these results provide the first evidence for a sequential recruitment of different MBD proteins and repressor complexes at distinct transitional stages of neuronal differentiation.

Fine motor skills are learned through repetitive practice and once learned, last for a long time. Skilled reaching is linked to structural and functional changes in multiple brain regions including, in particular, the primary motor cortex. Previous studies demonstrated that fine motor skill learning is associated with cortical synaptogenesis and motor map reorganization. At present, studies have implicated an indispensable role of epigenetic alterations in both hippocampal- and striatal-dependent memory formations, while examinations into the epigenetic changes in the primary motor cortex are lacking. The current study was aimed to identify epigenetic changes in motor cortex as a result of extensive motor skill learning using the single pellet skilled reaching task. Male Wistar rats were trained in the single pellet skilled reaching task (n = 6) for 10 consecutive days or were, under similar conditions, given access to pellets that did not require skilled reaches (n = 6). Skilled motor trained rats exhibited a rapid increase in successful reaches during the first four days of motor training before reaching a plateau, indicative of the acquisition and consolidation of the learned task, respectively. Expression profiles of chromatin modifying enzymes were screened using epigenetic-targeted PCR arrays. Results suggest that gene expression levels of multiple chromatin regulatory enzymes were down-regulated in the motor cortex of trained animals compared to controls following 10 days of motor training in the skilled reaching task. Among the chromatin modifying enzymes, the transcription level of Smyd1 (SET and MYND domain containing 1; NM_001106595) was lower (-2.17 fold-change) in motor cortex after 10 days of training compared to controls. Our results point to an epigenetic regulation of chromatin modification markers in the primary motor cortex that possibly underlie the mechanisms of synaptic plasticity, synaptogenesis and the formation of procedural memory.

The CTCF protein is involved in several important aspects of gene regulation including transcriptional activation, transcriptional repression and insulator ability. It is also involved in the regulation of epigenetic processes including X-chromosome inactivation and the maintenance of genomic imprinting. CTCF has been shown to bind to approximately 15 000 sites in the mammalian genome and has been implicated in nuclear organization. The CTCF protein mediates long-range chromatin interactions and is believed to form DNA loops. It also acts to block the communication of an enhancer with a promoter by acting as an insulator. Despite its importance in gene regulation, the molecular mechanisms that govern CTCF’s ability to perform its myriad functions remain enigmatic. In this thesis, I add insight into our understanding of the mechanisms behind CTCF’s function. I show that CTCF is post-translationally modified by the Small Ubiquitin-like Modifier proteins and that this post-translational modification contributes to its repressive ability at the c-myc P2 promoter. I also show that CTCF is localized to the sub-nuclear compartment called the Polycomb bodies. The Polycomb protein Pc2 acts as an E3 ligase to enhance the SUMOylation of CTCF by SUMOs 2 and 3. These findings help to explain CTCF’s ability to act as a transcriptional repressor. I also report biochemical evidence to support the role for CTCF in forming an unusual DNA structure, possibly a loop. I hypothesize that a single CTCF binding site is able to form DNA loops. These findings suggest mechanisms by which CTCF is able to organize the mammalian genome and to function as an insulator protein. In addition to these findings I have also purified CTCF interacting proteins through the use of the tandem affinity purification technique. The interacting proteins contain many chromatin and DNA binding proteins further suggesting a role for CTCF in chromatin organization. The results in this thesis enhance our knowledge of the molecular mechanisms of CTCF function and provide a basis for the improved understanding of CTCF mediated gene expression.

The CTCF protein is involved in several important aspects of gene regulation including transcriptional activation, transcriptional repression and insulator ability. It is also involved in the regulation of epigenetic processes including X-chromosome inactivation and the maintenance of genomic imprinting. CTCF has been shown to bind to approximately 15 000 sites in the mammalian genome and has been implicated in nuclear organization. The CTCF protein mediates long-range chromatin interactions and is believed to form DNA loops. It also acts to block the communication of an enhancer with a promoter by acting as an insulator. Despite its importance in gene regulation, the molecular mechanisms that govern CTCF’s ability to perform its myriad functions remain enigmatic. In this thesis, I add insight into our understanding of the mechanisms behind CTCF’s function. I show that CTCF is post-translationally modified by the Small Ubiquitin-like Modifier proteins and that this post-translational modification contributes to its repressive ability at the c-myc P2 promoter. I also show that CTCF is localized to the sub-nuclear compartment called the Polycomb bodies. The Polycomb protein Pc2 acts as an E3 ligase to enhance the SUMOylation of CTCF by SUMOs 2 and 3. These findings help to explain CTCF’s ability to act as a transcriptional repressor. I also report biochemical evidence to support the role for CTCF in forming an unusual DNA structure, possibly a loop. I hypothesize that a single CTCF binding site is able to form DNA loops. These findings suggest mechanisms by which CTCF is able to organize the mammalian genome and to function as an insulator protein. In addition to these findings I have also purified CTCF interacting proteins through the use of the tandem affinity purification technique. The interacting proteins contain many chromatin and DNA binding proteins further suggesting a role for CTCF in chromatin organization. The results in this thesis enhance our knowledge of the molecular mechanisms of CTCF function and provide a basis for the improved understanding of CTCF mediated gene expression.

Pregnane X receptor (PXR) is a ligand-dependent transcription factor that plays an important role in xenobiotic/drug metabolism. The ligand-receptor interaction transcriptionally activates phase I and phase II enzymes, and membrane-bound transporters in a coordinated manner and ultimately leads to detoxification and excretion of the ligands. One of the direct target genes is cytochrome P450 3A4 (CYP3A4) which is responsible for metabolism of over 50% of clinically used drugs. Understanding the regulation of PXR is important for treatment of disease and avoidance of untoward drug-drug interactions. In this research, we have used various biochemical and molecular approaches to investigate factors that regulate the transcriptional activity of PXR. We have stably transfected PXR into HepG2 human liver hepatoma cells. Using these PXR-HepG2 cells, we surveyed the histone methyltransferases that interact with PXR. Based on results from co-immunoprecipitation/methyltransferase, N-terminal peptide sequencing, GST-pulldown assays, we found that protein arginine methyltransferase 1 (PRMT1) is a predominant histone methyltransferase in HepG2 cells. Evidence from other laboratories suggests that histone methylation by PRMT1 sets the stage for subsequent histone modifications such as the acetylation of histone H4. These modifications are believed to be important for transcriptional and epigenetic regulation of gene expression. We hypothesize that PRMT1 plays a role in the epigenetic changes regulated by PXR. PRMT1-dependent histone methylation changes may be involved in epigenetic cell memory where prior exposure to certain agents may alter the chromatin (or priming the chromatin) with a "primed" state which alters the subsequent magnitude or duration of gene expression. In our study, we have found that pretreatment of PXR-HepG2 cells with DMSO greatly enhanced PXR-mediated activation of CYP3A4 upon rifampicin treatment. DMSO pretreatment altered histone modifications association with the promoter of the PXR-regulated gene (CYP3A4). Inhibition of histone methylation by PRMT1 either through RNAi or the methyltransferase inhibitor (Adox) abolished the priming effects. My research results strongly indicate that PRMT1 is involved in transcriptional regulation of PXR and may be involved in epigenetic memory of liver cells where prior exposure to agents changes the subsequent detoxification responses.

Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos. Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.

Ischaemic stroke rates are expected to rise significantly in the next decades due to an aging population. This increases the demand for new stroke biomarkers for early detection of patients at risk and new targets for treatment. It has been hypothesized that epigenetics may be important in the aetiology of stroke. The study consisted of three types of investigation: analysis of candidate gene polymorphism, candidate gene methylation analysis and epigenome-wide methylation analysis (EWAS) of pooled stroke and control samples. The stroke types studied were large vessel disease (LVD), small vessel disease (SVD) and cardioembolic stroke (CE). DNA from peripheral blood samples was used for EWAS and methylation analysis. Significant increases in rare allele frequency were observed in the EHMT2 and DNMT3B genes for all stroke cases; MBD2, DNMT3B and DNMT3L polymorphisms were associated with LVD. IL10, SOD3, LINE1 and PITX2 were significantly hypomethylated in LVD. IL10 and ALOX15 were hypomethylated in CE compared to controls. Methylation levels of following genes were associated with age (LINE1, IL10, MTHFR, TNFα, and PITX2), gender (SOD3 and LINE1), total cholesterol level (SOD3) and systolic blood pressure (IL10). HDAC9 genetic polymorphism was associated with the MTHFR methylation level. A distinctive methylation pattern for each stroke subtype was found by EWAS. The CE pool was hypomethylated at genome, chromosome and gene level, while LVD and SVD pools had regions with higher and lower methylation levels compared to the controls. GNAS was identified as new candidate gene by EWAS. The results suggested that genetic polymorphism and DNA methylation levels of candidate genes were associated with ischaemic stroke. Stroke subtypes had distinct methylation profiles suggesting differences in underlying aetiology. Variations in methylation levels detected in this study could lead to identification of specific biomarkers. Replication on a large number of subjects is required before final conclusions can be drawn.

No description available.

Paramutation is an allele interaction that causes a heritable change in the expression of one allele. At the b1 locus an interaction between B' and B-I alleles results in a change of B-I to B', symbolized by B'*. A combination of fine-structure mapping and transgenic approaches have demonstrated that the tandem repeats located ~100 kb upstream of the b1 transcription start site are sufficient for both paramutation and high expression.Plants carrying transgenes with tandem repeats in ectopic locations (repeat-transgene) were able to change B-I into B'*. The B'* state induced by the repeat-transgene was heritable and paramutagenic when segregated from the repeat-transgene. In addition, the repeat-transgene induced B-I silencing was prevented by the trans-acting mutation required for paramutation mop1-1, which was recently found to encode a RNA-dependent RNA polymerase (RdRP). Transgenes containing seven tandem repeats of only the 5' half of the sequence were able to paramutate B-I. Taken together, these results demonstrate that the paramutation sequences are contained in the 5' half of the repeats and they can paramutate B-I from non-allelic positions. Because paramutation induced by the repeat-transgenes and the endogenous B' allele are both heritable and depend on a functional RdRP, they likely involve a similar mechanism of RNA-mediated chromatin modification.Furthermore, we found that the tandem repeats are also sufficient for high expression of the b1 gene. When fused to a GUS reporter gene and introduced into maize, the tandem repeats enhanced GUS expression above the level observed for GUS transgenes that did not have the repeats. As observed with the endogenous B-I allele, the enhancer function of the repeats in the GUS transgenes is silenced by B' and the paramutagenic repeat-transgenes. After being with B' or the paramutagenic repeat-transgenes the repeats in the GUS constructs lost their ability to enhance gene expression.The identification of the tandem repeats as the sequences mediating paramutation suggest a new function for tandem repeats, mediating trans-interactions to establish heritable epigenetic states. Models are discussed for how alleles might communicate in trans to establish different epigenetic states and how the epigenetic state is maintained through mitosis and meiosis.

In women, tumors of the breast remain the most frequently diagnosed malignancy and the second leading cause of cancer-related deaths. One of the hallmarks of carcinogenesis is the abnormal silencing of tumor supprsssor genes by both genetic and epigenetic alterations, leading to defects in cell-cycle control, DNA repair, apoptosis and cell adhesion. This dissertation focuses on the elucidation of the genetic and epigenetic mechanisms associated with tumor suppressor gene silencing in human epithelial breast tumor cells, and the development of pharmacologic strategies aimed at reversing these types of repression through gene therapy and chromatin remodeling. Desmocollin 3 (DSC3) and MASPIN are anti-metastatic tumor suppressor genes that are silenced in a large percentage of breast tumors via aberrant DNA hypermethylation and histone hypoacetylation of their promoters. DSC3 and MASPIN are also p53-target genes, requiring its transcriptional activation to promote normal expression levels, yet a significant fraction of breast tumor cell lines express mutant forms of p53. Adenoviral-mediated re-introduction of wild type (wt) p53 into mutant p53-expressing breast tumor cells resulted in significant up-regulation of DSC3 and MASPIN expression, although not to the levels seen in normal breast epithelial cells. Mechanistically, the addition of wt p53 to these tumor cells resulted in increased histone acetylation and enhanced chromatin accessibility of the DSC3 and MASPIN promoters, despite continued cytosine hypermethylation. Pre-treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) prior to wt p53 addition produced synergistic reactivation of both DSC3 and MASPIN in breast cancer cells, approaching their levels in normal mammary cells. However, 5-aza-CdR did not significantly reduce DNA methylation in many cases as originally theorized. Therefore, follow-up studies focused on the identification of alternative, novel mechanisms of 5-aza-CdR-mediated induction of epigenetically silenced genes, finding that it consistently reduced transcriptionally repressive histone H3 lysine 9 (K9) methylation levels in the promoter regions of both DSC3 and MASPIN in breast tumor cells, by mediating global decreases in the histone H3 K9 methyltransferase, G9A. In summary, these results clearly show that cancer treatments targeting both genetic and epigenetic facets of gene regulation may be a useful strategy towards the therapeutic transcriptional reprogramming of cancer cells.