Mesenchymal stem cells are more and more widely used in tissue engineering due to their pluripotency and no relative ethical problems. Traditional characterization techniques to detect mesenchymal stem cell states include flow cytometry, gene expressing profiling and immunohistochemistry. However, these methods can only provide transient and low level information from current RNA or protein levels about mesenchymal stem cells, which may cause problems when predicting the possible downstream lineages they will commit into. We have developed chromatin immunoprecipitation (ChIP)-based epigenetic technique to detect mesenchymal stem cell states. For the systems we tested, this epigenetic assessor successfully characterized cell state changes and gave similar results obtained from gene expression profiling or protein expression assay. This epigenetic technique can provide information about mesenchymal stem cells states from a more fundamental chromatin level, which is promising for predicting future lineages from current states.

DNA methylation serves as a major epigenetic modification crucial to the normal organismal development and the onset and progression of complex diseases such as cancer. Computational predictions for DNA methylation profiling serve multiple purposes. First, accurate predictions can contribute valuable information for speeding up genome-wide DNA methylation profiling so that experimental resources can be focused on a few selected while computational procedures are applied to the bulk of the genome. Second, computational predictions can extract functional features and construct useful models of DNA methylation based on existing data, and can therefore be used as an initial step toward quantitative identification of critical factors or pathways controlling DNA methylation patterns. Third, computational prediction of DNA methylation can provide benchmark data to calibrate DNA methylation profiling equipment and to consolidate profiling results from different equipments or techniques. This thesis is written based on our study on the computational analysis of the DNA methylation patterns of the human genome. Particularly, we have established computational models (1) to predict the methylation patterns of the CpG islands in normal conditions, and (2) to detect the CpG islands that are unmethylated in normal conditions but aberrantly methylated in cancer conditions. When evaluated using the CD4 lymphocyte data of Human Epigenome Project (HEP) data set based on bisulfite sequencing, our computational models for predicting the methylation status of CpG islands in the normal conditions can achieve a high accuracy of 93-94%, specificity of 94%, and sensitivity of 92-93%. And, when evaluated using the aberrant methylation data from the MethCancerDB database for aberrantly methylated genes in cancer, our models for detecting the CpG islands that are unmethylated in normal conditions but aberrantly methylated in colon or prostate cancer can achieve an accuracy of 92-93%, specificity of 98-99%, and sensitivity of 92-93%.

Breast cancer is the most common malignancy affecting women and the second leading cause of death among women in the United States. Alcohol consumption is one of the few modifiable risk factors for breast cancer development but the mechanism by which it contributes to mammary cancer development and progression remains unclear, although it has been suggested that estrogen is critical for this process. To determine if alcohol promotes mammary tumor development via the estrogen pathway, estrogen receptor alpha-negative (ER[alpha]-negative) MMTV-neu mice were treated with various doses of ethanol and activation of estrogen signaling was measured. Our results showed that alcohol consumption increased estrogen signaling activation, serum estrogen levels and, most interestingly, expression of ER[alpha] in tumor tissue in the ER[alpha]-negative mice. Several lines of evidence in literature suggest that ER[alpha] expression in ER[alpha]-negative cancer cells is inhibited through epigenetic regulation. Epigenetics is the study of heritable changes in gene expression caused by mechanisms other than DNA sequence changes. Thus, to determine whether alcohol may regulate ER[alpha] re-expression in ER[alpha]-negative breast cancer cells through epigenetic mechanisms, we examined the effects of ethanol on CpG methylation and histone modifications (acetylation and methylation) of two ER[alpha]-negative breast cancer cell lines, MDA-MB-231 (human) and MMTV-neu (mouse). We also examined whether the epigenetic modifications subsequently affect the recruitment of transcriptional regulation complexes to the ER[alpha] promoter to regulate ER[alpha] transcription. Results showed that alcohol promotes ER[alpha] re-expression in these ER[alpha]-negative cell lines and that this effect was associated with decreased CpG methylation, an overall increase of histone acetylation and decrease of histone methylation, and an alteration in the enrichment of the ER[alpha] transcriptional regulation complexes (pRb2/p130-E2F4/5-HDAC1-SUV39H1-p300 and pRb2/p130-E2F4/5-HDAC1-SUV39H1-DNMT1) at the ER[alpha] promoter, which may contribute to cancer cell progression. In addition, we found that the inhibition of ER[alpha] by tamoxifen specifically blocks the effects of alcohol on ER[alpha] reactivation. To determine how alcohol promotes cell invasive ability, a critical process for cancer progression, we examined the role of two genes, metastasis suppressor Nm23 and integrin alpha-5 ITGA5, which we identified to be important for alcohol-induced breast cancer cell invasion. It has previously been shown that estrogen may regulate Nm23 expression and that estrogen regulation may be important for ITGA5-mediated cancer progression. Our results showed that alcohol promotes cancer cell invasion through the down-regulation of Nm23, which led to the subsequent increase of ITGA5 and increase of cell invasion. Collectively, data from my research strongly supports and provides evidence that alcohol promotes breast cancer development and progression through the regulation of estrogen signaling. / text

Out of the growing body of evidence demonstrating the role of higher-order chromatin organization within the nucleus in regulating the functions of the linear sequence of DNA emerges the genome as a physical entity. DNA packs into hierarchical levels of chromatin condensation, which then tailor accessibility to the linear sequence for nuclear processes while also serving as a central feature of nuclear organization. Further, varying condensation state alters the physical properties of the chromatin fiber. These may then exert or facilitate forces aiding in the spatial organization within the nucleus. Yet, this complex concept of nuclear structure even neglects the dynamic aspects of the genome continuously fluctuating and undergoing structural remodeling within the nucleus. Thus, while chromatin position within the nucleus is critical for biological functions including transcription, we must reconcile a particular position of a gene locus with the dynamic and physical nature of chromatin. Here we characterize the physical aspects of the genome associated with its dynamic properties that aid in regulation. We focus on developing techniques that measure the evolution of physical properties associated with nuclear processes. We leverage these techniques, capable of quantifying and spatially resolving its structural state within the nucleus and elucidating the underlying physics of its dynamics, to illuminate physical features associated with cellular processes. Specifically, we investigate the nuclear structural changes associated with growth factor stimulation on primary human cells known to impact large scale gene expression pathways. We also demonstrate dysfunction associated with these physical mechanisms accompany disease pathologies. Thus, we unify the biological understanding of cellular processes within the context of physical features of genome structure, organization and dynamics that are critical to human health and disease.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第19026号 / 農博第2104号 / 新制||農||1030 / 31977 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 今井 裕, 教授 久米 新一, 教授 松井 徹 / 学位規則第4条第1項該当

Inheritance of gene expression patterns through multiple rounds of cell division is crucial for the normal development of multi-cellular organisms and is mediated by epigenetic mechanisms. Many epigenetic mechanisms are believed to involve heritable changes to chromatin structure. This includes maintenance of transcriptional repression by Polycomb Group (PcG) proteins. It is unknown how PcG-dependent repression is maintained during or re-established after mitosis, a process that involves many physical and biochemical changes to chromatin. Understanding the behavior of PcG proteins during mitosis is key to answering this question: if PcG proteins remain bound in mitosis they may constitute the memory themselves, or else transcriptional memory must reside elsewhere, such as in the altered chromatin structures induced by PcG proteins. PcG protein association with chromosomes in mitosis in Drosophila S2 cells was examined by immunofluorescence and cellular fractionation. PcG proteins are associated with mitotic chromosomes, which is consistent with a role in carrying information about transcriptional repression through mitosis. Localization of PcG proteins to specific sites in the genome was assessed by chromatin immunoprecipitation (ChIP) followed by genome-wide sequencing (ChIP- SEQ) on mitotic cells. A method for isolating pure populations of mitotic cells was developed to access PcG protein localization in mitosis unambiguously. PcG proteins were not detected at well-characterized PcG targets including Hox genes on mitotic chromosomes, but a covalent modification of histone H3 associated with PcG- dependent repression, trimethylation of lysine 27 (H3K27me3), is retained at these sites. Two PcG proteins Posterior Sex Combs (PSC) and Polyhomeotic (PH) remain at about 10% of their interphase binding sites in mitosis. PSC and PH are preferentially retained in mitosis at sites that overlap recently described borders of chromatin domains (1), including sites that overlap domain borders flanking Hox gene clusters. These persistent binding sites may serve to nucleate re-establishment of PcG binding at target genes upon mitotic exit, perhaps with assistance of H3K27me3. Thus PcG proteins may form part of the transcriptional memory carried through mitosis, but perhaps not by persistent association at the targets of repression. Retention of elements at chromatin boundaries in mitosis may serve as a general mechanism for epigenetic memory.

No description available.

Medulloblastoma is a devastating disease of the cerebellum, and the most common solid pediatric malignancy of the central nervous system. Recently, transcriptome-wide profiling has dissected medulloblastoma from one single disease into four disparate molecular subgroups – namely WNT, SHH, Group3 and Group4. Distinct genomic, cytogenetic, mutational and clinical spectra associated with these subgroups highlight the pressing need for targeted therapies, of which encouraging preliminary results have been generated. While the promise of personalized medicine is within our reach, improved understanding of the molecular mechanisms driving pathogenesis is critical to this process. The intent of my PhD thesis research was to characterize the molecular mechanisms contributing to medulloblastoma pathogenesis, and the clinical impact of these aberrations. Through a combinatorial use of genetic and epigenetic profiling, next-generation sequencing and bioinformatics analyses we have identified subsets of tumors with transcriptional signatures that influence their clinical properties. Furthermore, our results have shed light on the establishment of the normal cerebellar cytoarchitecture, identifying a physiological glutamate gradient with critical implications to both cerebellar development and disease. This thesis stresses the importance of interrogating medulloblastoma in a subgroup-specific manner. Our findings demonstrate the utility of pursuing an integrated (copy number, mutational, transcriptional and epigenetic) molecular approach, to further our understanding of the pathobiology of medulloblastoma. Finally, we propose rationale therapeutic targets that may improve the treatment of aggressive variants of this disease.

Medulloblastoma is a devastating disease of the cerebellum, and the most common solid pediatric malignancy of the central nervous system. Recently, transcriptome-wide profiling has dissected medulloblastoma from one single disease into four disparate molecular subgroups – namely WNT, SHH, Group3 and Group4. Distinct genomic, cytogenetic, mutational and clinical spectra associated with these subgroups highlight the pressing need for targeted therapies, of which encouraging preliminary results have been generated. While the promise of personalized medicine is within our reach, improved understanding of the molecular mechanisms driving pathogenesis is critical to this process. The intent of my PhD thesis research was to characterize the molecular mechanisms contributing to medulloblastoma pathogenesis, and the clinical impact of these aberrations. Through a combinatorial use of genetic and epigenetic profiling, next-generation sequencing and bioinformatics analyses we have identified subsets of tumors with transcriptional signatures that influence their clinical properties. Furthermore, our results have shed light on the establishment of the normal cerebellar cytoarchitecture, identifying a physiological glutamate gradient with critical implications to both cerebellar development and disease. This thesis stresses the importance of interrogating medulloblastoma in a subgroup-specific manner. Our findings demonstrate the utility of pursuing an integrated (copy number, mutational, transcriptional and epigenetic) molecular approach, to further our understanding of the pathobiology of medulloblastoma. Finally, we propose rationale therapeutic targets that may improve the treatment of aggressive variants of this disease.

The highly developed and specialized anatomical and physiological characteristics observed for eukaryotes in general and mammals in particular are underwritten by an elaborate and intricate process of genome regulation. This precise control of the location, timing and amplitude of gene expression is achieved by a variety of genetic and epigenetic tools and mechanisms. While several of these regulatory mechanisms have been extensively studied, our understanding of the complex and diverse associations between various epigenetic marks and genetic elements with genome regulatory systems has remained incomplete. However, the recent profound improvements in sequencing technologies have significantly improved the depth and breadth to which their functions and relationships can be understood. The objective of this thesis has been to apply bioinformatics, computational and statistical tools to analyze and interpret various recent high throughput datasets from a combination of Next generation sequencing and Chromatin immune precipitation (ChIP-seq) experiments. These datasets have been analyzed to further our understanding of the dynamics of gene regulation in humans, particularly as it relates to repetitive DNA, cis-regulation and DNA methylation. The thesis thus resides at the intersection of three major areas; transposable elements, cis-regulatory elements and epigenetics. It explores how those three aspects of regulation relate with gene expression and the functional implications of those interactions. From this analysis, the thesis provides new insights into; 1) the relationship between the transposable element environment of human genes and their expression, 2) the role of mammalian-wide interspersed repeats (MIRs) in the function of human enhancers and enhancement of tissue-specic functions, 3) the existence and function of composite cis-regulatory elements and 4) the dynamics and relationship between human gene-body DNA methylation and gene expression.