Long terminal repeat (LTR) retrotransposons account for approximately 10% of mouse and 8% of human genomes and may play a role in modifying gene expression. Many species harbor retrotransposon families encompassing both autonomous and non-autonomous members. Specifically, the mouse Early Transposon (ETn) family members lack all retroviral genes but are transcriptionally and retrotranspositionally active, causing over 20 known insertional germline mutations. ETns owe their retrotransposition potential to proteins encoded by structurally intact MusD retrotransposons with whom they share LTRs. ETn elements are transcribed at a much higher level than MusD retrotransposons in embryos and undifferentiated cells, suggesting their evasion of host restriction mechanisms. However, mechanisms responsible for the replicative success of non-autonomous retrotransposon subfamilies over their coding-competent relatives are poorly understood. In the first stage of my research, I analyzed regulatory sequences in an ETn LTR responsible for its high promoter activity in the undifferentiated cell line P19. I found that three GC-boxes that may function as Sp1/Sp3 binding sites act synergistically and are indispensable for undifferentiated cell-specific promoter activity of the LTR. Sp1 binding partners may be responsible for the restricted ETn expression. Moreover, I have shown that unlike many retroviruses, ETn elements possess multiple transcription initiation sites and that they have amplified via intracellular retrotransposition in the P19 teratocarcinoma cell line. In the next step of my research, I performed analysis of epigenetic mechanisms as a means of ERV suppression. Specifically, I showed that in embryonic stem cells, autonomous MusD retrotransposons are epigenetically suppressed to a greater degree than non-autonomous ETn retrotransposons, illustrated by a higher level of DNA methylation and a lower level of active histone modifications. I hypothesize that MusD elements may be silenced by DNA methylation and repressive chromatin spreading into the LTR from the CpG-rich internal retroviral sequence absent in ETn elements. I propose that internal structure largely devoid of high CG content enables ETn elements to evade host-imposed transcriptional repression, contributing to their high mutagenic activity in the mouse germline.

Correlation between 8-hydroxy-2-deoxyguanosine (8-OHdG) and/or redox imbalance and 5-methylcytosine (5mc) levels was studied in patients with bipolar disorder (BD) and an excellent response to lithium treatment, their affected and unaffected relatives (AR and UR, respectively) and controls. We demonstrated that global methylation was decreased in BD subjects and their relatives compared to controls and remained so after lithium treatment in BD subjects, while UR and AR were no longer different than controls. 8-OHdG levels and Complex I activity did not differ between groups before and after lithium treatment. Finally, glutathione peroxidase activity was higher in relatives of patients before and after lithium treatment and negatively correlated with 5mc levels. The methylation pattern in BD subjects may be specific for BD and that lithium may be involved in regulating global methylation. This study supports the antioxidant properties of lithium through regulation of glutathione system.

Correlation between 8-hydroxy-2-deoxyguanosine (8-OHdG) and/or redox imbalance and 5-methylcytosine (5mc) levels was studied in patients with bipolar disorder (BD) and an excellent response to lithium treatment, their affected and unaffected relatives (AR and UR, respectively) and controls. We demonstrated that global methylation was decreased in BD subjects and their relatives compared to controls and remained so after lithium treatment in BD subjects, while UR and AR were no longer different than controls. 8-OHdG levels and Complex I activity did not differ between groups before and after lithium treatment. Finally, glutathione peroxidase activity was higher in relatives of patients before and after lithium treatment and negatively correlated with 5mc levels. The methylation pattern in BD subjects may be specific for BD and that lithium may be involved in regulating global methylation. This study supports the antioxidant properties of lithium through regulation of glutathione system.

DNA replication is cruciaI for the transmission of genetic information. Understanding the enzymology involved in this complex process will allow further insight into its mechanism. Experimental evidence indicates a role for DNA ligase I in DNA replication. Techniques of molecular and cellular biology and immunology were utilized in this study to further investigate DNA ligase I and clarify its involvement and interaction with other proteins in DNA replication. Immunofluorescence studies were performed to examine the intracellular distribution of DNA ligase I. Confocal analysis of indirect immunofluorescence detection of DNA ligase I using affinity purified anti-human DNA ligase I antibodies showed nuclear localization of DNA ligase I in distinct foci resembling those structures seen in detection of centres of DNA replication and other DNA replication proteins. Immunoprecipitation experiments were performed on extracts of MDBK cells to examine possible interactions of DNA ligase I with the DNA replication cofactor, PCNA; and no interactions were detected.

Monozygotic (MZ) twins are currently used in epigenetic studies as one homogenous group. However, there are two MZ twin types: dichorionic (DC) and monochorionic (MC). We hypothesize that DCMZ twins are more similar epigenetically, compared to MCMZ twins, due to earlier zygote splitting, a higher degree of birth outcome similarity, or both. We recruited 220 newborn twins and obtained genome-wide DNA methylation profiles for 48 twins. Intraclass correlation coefficients (ICC) and linear mixed models were used to investigate the relationships between DNA methylation levels and chorionicity. DCMZ twins tended to have longer gestational age, larger birth weight, and smaller birth length discordance. DCMZ twins had more similar DNA methylation profiles than MCMZ twins (ICC=0.21 vs. 0.13), after adjusting for birth outcomes. Additionally, we identified 5,170 CpG sites with different DNA methylation levels between DCMZ and MCMZ twins. This study highlights the importance of incorporating chorionicity information in epigenetic twin studies.

Ascorbic acid (AA), in popular speech vitamin C, is a commonly known nutrient. It is involved in several biological processes and deficiency can lead to scurvy. Recent publications have shown the impact of AA on epigenetic regulation in mice. Addition of AA, via enzymatic activity, enhances the generation of 5-hydroxymethylcytosine (5hmC), which is an intermediate in active demethylation of DNA. The role of AA on epigenetic changes in humans has to our knowledge never been studied. In this study, naïve CD4+ T cells from blood donors were used as a model system to investigate AAs possible role in methylation changes in the immune system. By using dot-blot assay, hydroxymethylated DNA immunoprecipitation (hmeDIP) and qPCR, changes in methylation executed by AA could be detected. A confirmation of AAs impact on epigenetic changes in mice was observed. AA enhanced the levels of 5hmC compared to untreated cells. The Jurkat cell line, a human T lymphocyte cell line, showed an opposite result. Treatment with AA decreased the levels of 5hmC compared to untreated cells. When comparing this result with the results obtained in human naïve T cells, the same observation was made. The difference between mouse and human in the ability of producing and metabolize AA could be a reason for this opposite result. Since AA had the ability to modify epigenetic changes in primary human CD4+ T cells, the results suggest that AA may have a function in the human immune system.

DNA methylation has been shown to be involved in switching a number of genes on or off in particular cells. The relationship between DNA methylation and $ beta$-casein gene expression in the mammary tissue of lactating cows and mammary epithelial cells was examined. A positive correlation existed between hypomethylation of two MspI/HpaII sites in the body and one MspI/HpaII site in the 3$ sp prime$ end of the $ beta$-casein gene and its expression. In addition to these sites, hypomethylation of a distal MspI/HpaII site and HindIII sensitivity at a HindIII site also correlated with gene expression. Five DNase I hypersensitive sites were located within a 8 kb fragment. These sites designated as H1 to H5 were mapped approximately $-5, -1.3, -0.2,$ 1.7 and 2.5 kb with respect to the start site of transcription, respectively. The H2 and H3 sites were within a 1790 bp sequence that has been reported to contain a responsive element for prolactin and extracellular matrix dependent regulation and the binding site for mammary gland specific factor. / To study the dynamic changes in hypomenthylation at the MspI/HpaII sites and HindIII sensitivity, mammary tissues from pregnant heifers were evaluated. Site specific demethylation was observed depending on the stage of gestation. Demethylation of two MspI/HpaII sites (denoted M2 and M4) occurred during the early gestation, progressed slowly until mid-pregnancy, and rapidly during the last part of pregnancy. During the early stages of gestation, changes in the HindIII sensitivity in the coding domain of the $ beta$-casein gene also took place. Despite changes in HindIII sensitivity, the second HindIII site remained resistant to HindIII. By the fifth stage of gestation, the third MspI/HpaII site (M3) became less methylated and during this time the H2 site became more sensitive to HindIII. Northern analysis confirmed that demethylation of the M3 site and the acquisition of HindIII sensitivity at the H2 site was correlated with $ beta$-casein transcription. / Although $ alpha$-lactalbumin and $ beta$-casein genes are structurally and evolutionarily unrelated, they likely share common regulatory features, since both are expressed in the mammary gland during lactation. To investigate this possibility, methylation of the $ alpha$-lactalbumin gene was examined. In vivo studies revealed hypomethylation of the bovine $ alpha$-lactalbumin gene at two MspI sites and a cluster of two HhaI sites during the first and second stage of gestation, respectively. Furthermore, hypomethylation events occured only in the functional gene and not in pseudogenes, and the hypomethylation pattern was established prior to gene expression. / Taken together, the present finding suggest that DNA hypomethylation is necessary for the expression of two mammary-specific milk protein genes, $ beta$-casein and $ alpha$-lactalbumin. Hypermethylation within the body of these genes may silence these genes in non-expressing tissues and in non-epithelial cells within the mammary gland during lactation.

This study examined the DNA replication timing patterns of endothelial cell (EC)-enriched genes. We especially focused on a unique set of EC-enriched mRNA transcripts that possess differentially methylated regions (DMRs) within proximal promoters. It was previously shown that this DNA methylation plays an important functional role in regulating EC-enriched patterns of gene expression. Since the maintenance of these silencing marks is necessary for the inheritance of cell identity, the cell should ensure the proper transmission of such marks during mitotic cell cycle. Here we show that EC-enriched genes with DMRs replicate early during S phase in both expressing and non-expressing cell types. EC-enriched genes that do not have DMRs followed the expected trend, being early replicating in expressing cell types and late in non-expressing cell types. The relationship between DNA replication and DNA methylation was also investigated. A delay between DNA replication and DNA methylation was observed.

Aberrant DNA methylation in gene promoters causes gene silencing and is a common event in prostate cancer development and progression. While commonly identified methylated genes have been analyzed for their potential clinical utility in a variety of cancers, few studies have attempted a genome-wide methylation approach to discover new and possibly improved biomarkers for prostate cancer. In order to identify DNA methylation changes associated with aggressive prostate cancer, we performed a genome-wide analysis of 40 prostate cancers using Agilent human CpG island microarrays. Methylation profiles of candidate genes were validated using quantitative MethyLight technology in an independent series of 219 radical prostatectomies and compared to clinicopathological parameters. The effects of methylation on expression of HOXD3 and HOXD8 and the possible role of HOXD8 in progression of PCa were also investigated. We discovered previously unidentified methylation in the HOXD cluster of genes, namely HOXD3 and HOXD8, as well as TGFβ2 and GENE X as potential prognostic biomarkers. Furthermore, unsupervised clustering of samples by methylation signature indicated ERG oncogene expression as significantly different between clusters. Within the independent cohort, we observed strong correlations between Gleason score (GS) and HOXD3 as well as GENE X, while HOXD3 and HOXD8 methylation were associated with ERG expresson. TGFβ2 was an independent predictor of disease recurrence using Cox multivariate regression analysis. In gene expression studies, both HOXD3 and HOXD8 were elevated in cancers with poor prognosis, while DNA methylation did not correlate with expression levels. Both genes were found to contain alternative transcription start sites, explaining the poor correlation between methylation and expression. Finally, knockdown of HOXD8 expression did not have any effect on viable cells or cell motility in an in vitro model. These results indicate that a panel of novel DNA methylation markers distinguish indolent prostate cancers from aggressive ones, and that expression of HOXD3 and HOXD8 is regulated by mechanisms including, but not dependent on, DNA methylation.

This study examined the DNA replication timing patterns of endothelial cell (EC)-enriched genes. We especially focused on a unique set of EC-enriched mRNA transcripts that possess differentially methylated regions (DMRs) within proximal promoters. It was previously shown that this DNA methylation plays an important functional role in regulating EC-enriched patterns of gene expression. Since the maintenance of these silencing marks is necessary for the inheritance of cell identity, the cell should ensure the proper transmission of such marks during mitotic cell cycle. Here we show that EC-enriched genes with DMRs replicate early during S phase in both expressing and non-expressing cell types. EC-enriched genes that do not have DMRs followed the expected trend, being early replicating in expressing cell types and late in non-expressing cell types. The relationship between DNA replication and DNA methylation was also investigated. A delay between DNA replication and DNA methylation was observed.