Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

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The family of transcription factors TEAD regulates the expression of genes that affect cell proliferation, differentiation and apoptosis. Activity of TEAD1 is regulated via the Hippo signaling pathway. General mechanism of tumor cell suppression by the Hippo signaling pathway remains unclear. C-MYC and GLUT1, the two key regulators of glycolysis, were recently described as targets of the Hippo signaling pathway in human leukemia cells. In this diploma thesis, the interaction of TEAD1 with M-CAT binding motifs was experimentally confirmed in the first exon of C-MYC gene. In addition, a new interaction of TEAD1 with M-CAT binding motifs has been found in the enhancer of C-MYC promoter and enhancer of GLUT1 promoter by ChIP analysis. Regulation of glucose metabolism by the Hippo signaling pathway may represent a new mechanism of tumor cell suppression. Key words: Gene regulation, transcription factors, chromatin immunoprecipitation, bioinformatics

The TEAD family of transcription factors regulates expression of genes affecting cell proliferation, differentiation and apoptosis. The activity of a particular transcription factor called TEAD1 is regulated by the Hippo signalling pathway. The Hippo pathway has been implicated to play a role in cancer suppression, however its precise mechanism remains unclear. MYC and GLUT1, genes which are coding two key regulators of glycolysis, were recently described as potential targets of the Hippo signalling pathway in human leukemia cells. In this diploma thesis, I tried to confirm the proposed interaction of the transcription factor TEAD1 with regulatory sequences of MYC and GLUT1 genes using chromatin immunoprecipitation (ChIP) analysis in human leukemic cells. However, I failed to successfully isolate TEAD1 complexes using ChIP. So, I discuss in my diploma thesis also possible reasons for this outcome, including biological and methodological issues. (In Czech) Key words: Transcriptional regulation, TEAD transcription factors, chromatin immunoprecipitation, leukemia

Covalent modifications of histones play a critical role in many important biological processes such as transcription, DNA repair and recombination. Among the major modifications known so far, histone H3 acetylation at lysines 9 and 14 (H3K9/14), monoubiquitination of histone H2B at lysine123 (H2BK123) and H3 lysine 4 methylation (H3K4) are among the more studied ones. The importances of these modifications have been further stressed by its connection to various human diseases including cancers. Previous biochemical studies have shown that H2BK123 ubiquitination is mandatory for methylation at histone H3K4. However, little is known about the regulatory mechanisms of H3K4 methylation by H2B ubiquitination in vivo. Thus, the prime focus of this study is to understand the factors involved in the regulation of H2B ubiquitination, the regulatory mechanisms of the cross-talk between H2BK123 ubiquitination and H3K4 methylation and the role of these covalent modifications in transcriptional regulation under physiological conditions. Here in this study, I have shown that Ubp8p, a histone deubiquitinase, is a bona fide subunit of SAGA (Spt3-Ada-Gcn5 acetyltransferase) co-activator complex and selectively regulates both di and trimethylation of histone H3K4 at the core promoter of a SAGA-dependent gene in vivo. However, over the open reading frames for a subset of constitutive genes H2B ubiquitination selectively upregulates only H3K4 trimethylation but not dimethylation. Moreover, such an upregulation of H3K4 trimethylation has no impact on the RNA Polymerase II (RNAPII) recruitment and hence transcription of the respective genes. Interestingly, at an inducible gene, histone H2B ubiquitination promotes transcription elongation independently of H3K4 methylation. Furthermore, this study also demonstrates for the first time, the molecular mechanism for the cross-talk between H2B ubiquitination and H3K4 methylation in vivo. Evidently a COMPASS subunit, Cps35p, is necessary for the trans-tail cross talk between histones H2B and H3. Finally, this study also shows that Sgf73p, a SAGA subunit, is required for SAGA recruitment at the promoters of several SAGA dependent genes and facilitates transcription in both HAT-dependent and HAT-independent manner. Collectively, the results from this study not only provide deep insights into the regulatory mechanisms of H2B ubiquitination and H3K4 methylation (and their role in transcription) but also give a new functional dimension to SAGA subunit, Sgf73p, under physiological conditions. Given the role of histone acetylation, ubiquitination and methylation in many human diseases, the results from this study is of tremendous clinical value unveiling new therapeutical targets.

Ocean warming is leading to increased occurrence of coral mass bleaching events, threatening the persistence of these ecosystems and the communities that rely on them. While reef recovery is possible, conservation approaches based purely on transplantation/coral-gardening will not suffice to maintain these ecosystems over the projected environmental changes. Assisted evolution approaches aim to boost acclimatization and adaptation processes. A potential approach could be to harness the naturally occurring mechanism of environmental memory that has been observed in corals and other organisms, where an organism remembers a priming stress event to allow a faster/stronger response when the stress re-occurs. In this thesis I aimed to investigate whether this mechanism exists and how it is regulated on a molecular level in the sea anemone Aiptasia. Aiptasia were primed to heat stress by exposing them to 32 °C water for several years, or for one week. After a recovery period of one week at 25 °C, a naïve and the primed treatments were exposed to lethal thermal stress at 34 °C for three days. Primed treatments performed better than the naïve treatment in survival, photosynthetic efficiency and symbiont density for two days, after which the priming advantage was lost. The difference between the primed treatments indicated that the priming dose may affect priming success. There were clear indications of an epigenetic transcriptional memory mechanism on a transcriptional level. I observed a pronounced difference between control and heat-stressed treatments, indicating that transcription returned to near baseline expression after cessation of the priming exposure. The functional categories of differentially expressed genes in heat stress relative to control were similar between naïve and primed treatments, with the main difference observed in a stronger up- and downregulation of stress response genes in the long-term primed treatment. I optimized a chromatin immunoprecipitation protocol for use with Aiptasia by adjusting fixation, sonication and immunoprecipitation conditions. The enrichment of H3K4me2/me3 and poised RNA Pol II in the promoters of stress response genes will be investigated next to elucidate the mechanism of the observed epigenetic transcriptional memory in Aiptasia, and to ultimately inform conservation strategies for coral reefs globally.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13517号 / 論医博第2267号 / 新制||医||1061(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 遊佐 宏介, 教授 戸井 雅和, 教授 小川 誠司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Previous studies have shown that genes can be switched on or off by age, environmental factors, diseases, and lifestyles. The open or compact structures of chromatin is a crucial factor that affects gene expression. Epigenetics refers to hereditary mechanisms that change gene expression and regulations without changing DNA sequences. Epigenetic modifications, such as DNA methylation, histone modification, and non-coding RNA interaction, play critical roles in cell differentiation and disease processes. The conventional approach requires the use of a few million or more cells as starting material. However, such quantity is not available when samples from patients and small lab animals are examined. Microfluidic technology offers advantages to utilize low-input starting material and for high-throughput. In this thesis, I developed novel microfluidic technologies to study epigenomic regulations, including 1) profiling epigenomic changes associated with LPS-induced murine monocytes for immunotherapy, 2) examining cell-type-specific epigenomic changes associated with BRCA1 mutation in breast tissues for breast cancer treatment, and 3) developing a novel microfluidic oscillatory hybridized ChIRP-seq assay to profile genome-wide lncRNA binding for numerous human diseases. We used 20,000 and 50,000 primary cells to study histone modifications in inflammation and breast cancer of BRCA1 mutation, respectively. In the project of whole-genome lncRNA bindings, our microfluidic ChIRP-seq assay, for the first time, allowed us to probe native lncRNA bindings in mouse tissue samples successfully. The technology is a promising approach for scientists to study lncRNA bindings in primary patients. Our works pave the way for low-input and high-throughput epigenomic profiling for precision medicine development. / Doctor of Philosophy / Traditionally, physicians treat patients with a one-size-fits-all approach, in which disease prevention and treatment are designed for the average person. The one-size-fits-all approach fits many patients, but does not work on some. Precision medicine is launched to improve the low efficiency and diminish side effects, and all of these drawbacks are happening in the traditional approaches. The genomic, transcriptomic, and epigenomic data from patients is a valuable resource for developing precision medicine. Conventional approaches in profiling functional epigenomic regulation use tens to hundreds of millions cells per assay, that is why applications in clinical samples are restricted for several decades. Due to the small volume manipulated in microfluidic devices, microfluidic technology exhibits high efficiency in easy operation, reducing the required number of cells, and improving the sensitivity of assays. In order to examine functional epigenomic regulations, we developed novel microfluidic technologies for applications with the small number of cells. We used 20,000 cells from mice to study the epigenomic changes in monocytes. We also used 50,000 cells from patients and mice to study epigenomic changes associated with BRCA1 mutation in different cell types. We developed a novel microfluidic technology for studying lncRNA bindings. We used 100,000-500,000 cells from cell lines and primary tissues to test several lncRNAs. Traditional approaches require 20-100 million cells per assay, and these cells are infected by virus for over-producing specific lncRNA. However, our technology just needs 100,000 cells (non-over-producing state) to study lncRNA bindings. To the best of our knowledge, this is the first allowed us to study native lncRNA bindings in mouse samples successfully. Our efforts in developing microfluidic technologies and studying epigenomic regulations pave the way for precision medicine development.

Current sequencing technologies allows researchers unprecedented insight into our biology, and how these biological mechanisms can become distorted and lead to disease. These aberrant mechanisms can be brought about by many causes, but some occur as a result of genetic mutations or external factors through the epigenome. Here, we used our microfluidic technology to profile the epigenome and transcriptome to study such aberrant mechanisms in three different diseases and illnesses: breast cancer, chronic inflammation, and mental illness. We profiled the epigenome of breast tissue from healthy women with the BRCA1 mutation to understand how the mutation may facilitate eventual breast cancer. Epigenomic changes in breast cells suggest that cells in the basal compartment may differentiate into a different cell type, and perhaps become the source of breast cancer. Next, we compared the epigenome and genome of murine immune cells under low-grade inflammation and acute inflammation conditions. We found that low-grade inflammation preferentially utilizes different signaling pathways than in acute inflammation, and this may lead to a non-resolving state. Finally, we analyzed the effect of the maternal immune activation on unborn offspring, and how these changes could cause later mental illness. The insights we made into these diseases may lead to future therapies. / Doctor of Philosophy / Despite advances in medical and scientific research, there is still a dearth of information on how diseases affect the expression of our genes, such as breast cancer, chronic inflammation, and influenza. Mutation in the BRCA1 gene is probably the most well-known mutation that can lead to breast cancer. We know the overarching reason that mutation in BRCA1 can lead to cancer, as BRCA1 is responsible for repairing damage in the DNA, so mutations can compound and create cancerous cells. However, we do not know the exact mechanisms by which this actually happens. Another widespread problem is chronic inflammation, which can promote or lead to diseases such as diabetes, cancer, Alzheimer's, Rheumatoid arthritis, and heart disease. In addition, there are many causes of chronic inflammation that many people have experienced at some point in time, including stress, insomnia, being sedentary, poor eating habits, and obesity. Despite this, we still do not fully understand why chronic inflammation differs from normal inflammation, which is a healthy process, or why it does not resolve. There are also other connections that are surprising, and many are not aware of. If a pregnant woman gets the flu during her second trimester, her baby has much higher odds of developing schizophrenia later in its lifetime. Given the prevalence of the flu, there is a very real chance that an expecting mother will be infected during her pregnancy.