Indiana University-Purdue University Indianapolis (IUPUI) / A subpopulation of tumor cells known as ovarian cancer initiating cells (OCICs) have been shown to be the cells that propagate the tumor phenotype in ovarian cancer. Studies have showed that a very small population (100) of these cells is sufficient to induce a tumor phenotype; while a large quantity of tumor cells (5 X 105) are required to induce such a phenotype. In this study we studied the functional changes in genes expressed in the OCIC phenotype which were important for such efficient propagation of cancers. To enable this analysis, we generated mRNA expression and DNA methylation profiles of OCICs and compared them with those of tumor and normal ovarian surface epithelial cells. We identified four pathways which regulated most of the observed changes and were predicted to be important factors in distinguishing the OCICs from tumors and normal cells. The gene signatures for these pathways were analyzed by unsupervised clustering in order to determine the similarities of OCICs with respect to tumor and normal samples. We further believed that the OCICs can be used as indicators towards the genesis and progression of early events in the ovarian cancers. In light of this, we considered two hypotheses which are currently addressing the genesis of ovarian cancer. The first hypothesis proposed ovarian surface epithelial cells to be cells of origin of the ovarian cancer while the other proposed the fallopian tube cells to be contributing the cell of origin for these cancers. It is also believed that these two cells can be reciprocal cells of origin for the cancer phenotype. In order to test these hypotheses, we integrated the in-house dataset with a public domain fallopian tube gene expression data. The integration of the results obtained from these analyses provided better understanding of the early events in ovarian carcinogenesis.

The molecular target of epigenetic signaling is chromatin. Histones are extensively post-translationally modified (PTM), and many of these individual modifications have been studied in depth. As PTMs occur at multiple positions within histones, the degree to which these modifications might influence each other remains one of the major challenges of chromatin biology. Although major discoveries in understanding the complex repertoire of histone modifications were achieved using reductionist experimental systems with synthetic histone peptides, they do not explain the role of putative PTM cross-talks in a chromatin context. However, generating chromatin substrates of defined modification status has proved to be a technically challenging task. In this thesis, I first demonstrate our work on establishing a novel approach to produce libraries of modified nucleosomes. We employed protein trans-splicing and sortase-mediated ligation strategies to incorporate chemical modifications on histone tails of ‘ligation-ready’ nucleosomes. Subsequently, the ‘designer’ nucleosome libraries were used for testing the binding of heterochromatin protein 1 (HP1) and elucidated the previously uncharacterized crosstalk of H3K9me2 and S28ph marks. Further investigations explained the mechanism of this crosstalk and highlighted the importance of developing chemical biology tools for elucidating complex chromatin signaling. Second, I describe our reconstitution systems for the assembly of semisynthetic recombinant chromatin carrying methylation marks on DNA and distinct modifications on histones, e.g. H3K9me3. I aimed to understand the mechanisms of the interplay between chromatin and one of the DNA maintenance methylation factors, UHRF1. I showed that UHRF1 strongly interacts with nucleosomes containing linker DNA. However, it exerts only residual enzymatic activity in this context. Based on functional H3 ubiquitylation assays in vitro, I found that hemi-methylated nucleosomes stimulate enzymatic activity of UHRF1, suggesting that the protein’s chromatin targeting and activation are a two-step process. The positioning of hemi-methylated CpG on nucleosome regulates UHRF1 target selectivity. Further, mutational analysis revealed that the PHD domain of the factor is indispensable for H3 binding and that its SRA domain is required for catalytic activation. Overall, our work adds a new layer of positional complexity to the me½CpG-dependent regulation of UHRF1 and expands the current model of DNA methylation maintenance.

Polycyclic aromatic hydrocarbons (PAHs) comprise an important class of environmental pollutants that are known to cause lung cancer in animals and suspected lung carcinogens in humans. PAHs are also known to cause cancer in offspring when provided to a pregnant mouse. Some evidence from cell-based studies points to PAHs as modulators of the epigenome, that is modifications to DNA structure that control the expression of genes. Inappropriate changes to the epigenome and consequently expression of cancer-critical genes are often characteristic of cancer cells. The objective of this thesis research was to determine the impact of transplacental exposure to two model PAHs on the epigenome of fetal and adult lung tissues in offspring. Specifically, we measured patterns of methylation of DNA, a type of epigenetic mark, in different types of lung tissue to assess changes in the epigenome associated with development of lung cancer. Two strategies were employed: 1) a targeted approach using ultra-deep bisulfite sequencing to precisely measure the specific pattern of methylated sites in the promoter regulatory region for several tumor suppressor genes, including Cdkn2a, Rarb, Dapk1 and Mgmt; and 2) a broad, genome-wide approach using a microarray covering all regulatory promoter regions in the entire mouse genome. Our first approach did not yield any marked differences in methylation patterns for any of the target genes for lung tissues obtained at birth or at various ages up to 45 weeks, nor according to the type of tissue (normal, pre-neoplastic, tumor). However, the genome-wide approach did yield specific patterns of methylation in lung tumors, including distinct profiles associated with lung tumor tissue from PAH-exposed animals that were substantially different from normal lung tissue in control animals. Altogether, the research presented here identified several new target genes of interest for future studies investigating the epigenetics of PAHinitiated lung cancer. This work also provided new knowledge that exposure to PAHs can lead to distinct DNA methylation profiles in lung tumors in adult offspring.

Indiana University-Purdue University Indianapolis (IUPUI)

The experiments presented were designed to test the hypothesis that the well-known carcinogen, benzo[a]pyrene has epigenetic effects, specifically the ability to alter cytosine methylation patterns. MCF-7 and MDA-MB-231 human breast cancer cells were treated for a period of sixty days with 100 nM benzo[a]pyrene. The methylation status of two genes, Ecadherin and GSTP 1 were examined using methyl-specific PCR and Southern blot analysis. After sixty days, no detectable change in methylation was observed. Evidence exists that de novo methylation is a consequence of transcriptional inactivity. Benzo[a]pyrene can contribute to transcriptional repression by sequestering the transcription factor, Spl. To test this hypothesis in our system, MCF-7 cells were transiently transfected with a reporter construct containing Sp 1 sites. These experiments demonstrated an 8.4 fold increase in reporter gene activity over a promoterless control plasmid; however, a difference could not be established between benzo[a]pyrene-treated and untreated cells.

http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path[]=8620&pubmed-linkout=1 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19927号 / 医博第4147号 / 新制||医||1017(附属図書館) / 33013 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 泰広, 教授 妹尾 浩, 教授 武田 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20272号 / 医博第4231号 / 新制||医||1021(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 泰広, 教授 藤渕 航, 教授 一山 智 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21023号 / 医科博第84号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 萩原 正敏, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Epigenetic marks encoded by DNA methylation, drifts with age, this results in gain of methylation in CpG islands and loss of methylation in repeat regions. This drift correlates with lifespan and is conserved across species mice, rhesus monkeys, and humans. However, the biology of how this drift occurs is still unexplained. Our hypothesis is that epigenetic drift occurs in aging stem cells. i.e. young stem cells have a stable, uniform DNA methylation pattern. However, as stem cells age, due to cell division and environmental effects, old stem cells have an unstable mosaic methylation pattern which leads to stem cell depletion and exhaustion, focal proliferation, etc., causing cancer and various age-related diseases. To evaluate this hypothesis, we analyzed DNA methylation pattern in mouse colon intestinal stem cells (Lgr5-gfp+) mice from four different age groups (4,12,18, and 24-months) against nonstem cells (Lgr5-GFP-). We found that there are not many methylation changes between stem cells and nonstem cells of the same age (0% change in 4 month and +- 0.2% in 24-month-old samples). This demonstrates that DNA methylation changes primarily occur in stem cells. Permutation analysis of all four aging time points (stem and nonstem) revealed 8102 CpG sites that changed methylation with age, with hypermethylated CpG sites in the promoter-nonCpG islands having the highest odds of gaining methylation with age. CpG sites that changed in DNA methylation levels between tissues (colon and small intestine) were also analyzed through permutation testing. CpG sites that changed between tissues did not overlap with age, showing that CpG sites that change with age and differentiation are different. Gene expression changes with aging was also analyzed, however, compared to DNA methylation not many genes changed in expression with age. We compared the correlation between levels of DNA methylation and gene expression. Low levels of methylation (0-10%) had higher levels of gene expression and increase in DNA methylation led to a decrease in RNA expression. We compared gene expression with CpG sites that change significantly with age in the promoter region from the permutation analysis and found that gene expression is correlated with DNA methylation in CpG sites in promoter-CpG island (r= -0.18). We used principles of entropy and information theory to better analyze the pattern of methylation drift with age in the intestine and found that entropy, measured through Jensen-Shannon Distance (JSD), increased with age but not with differentiation from stem to nonstem cells and the entropy increased 2.5-3 folds in the old when compared to the young. We extended this analysis to other organs with different rates of cell division and found that entropy does not change with age in organs with low rate of cell division such as the heart and kidney. Thus, the rate of cell division and change in entropy with age are highly correlated (r=0.89, p =<0.001). This demonstrates that we can use DNA methylation entropy to measure stem cell replication and aging. We also did not find many loci that changed with age overlapping across tissues, showing that change in methylation entropy is tissue specific. Thus, our data suggests that tissue specific clocks that measure entropy may provide a more accurate measurement of methylation age when compared to clocks based solely on percent methylation. Finally, we looked at DNA methylation changes in intestinal organoids and compared them to primary stem and nonstem cells and found a very weak correlation between them (r=0.23, p=<0.001) and no correlation as the organoids are passaged. We assessed DNA methylation changes caused by long-term passaging (28 weeks). DNA methylation changes with passage, in 4-month and 24-month upper small intestine (USI) organoids, was compared to aging in USI primary cells and we found a weak correlation with 4-month organoids (r=0.28, p=<0.001) and no correlation in 24-month organoids passaged long-term. This demonstrates there are changes in DNA methylation when transitioning from in vivo to in vitro and could result in gene expression and function of the organoid. More studies need to be done to assess the impact of these changes. / Biomedical Sciences