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

No description available.

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

No description available.

The main focus of this research was the development of microfluidic technology for ultrasensitive and fast molecular analysis of cells. Chromatin immunoprecipitation (ChIP) assay followed by next generation sequencing serves as the primary technique to characterize the genomic locations associated with histone modifications. However, conventional ChIP-seq assay requires large numbers of cells. We demonstrate a novel microfluidics-based ChIP-seq assay which dramatically reduced the required cell number. Coupled with next generation sequencing, the assay permitted the analysis of histone modifications at the whole genome from as few as ~100 cells. Using the same device, we demonstrated that MeDIP-seq with tiny amount of DNA (<5ng) generated high quality genome-wide profiles of DNA methylation. Off-chip sonication often leads to sample loss due to multiple tube transferring. In addition, conventional sonicators are not able to manipulate samples with small volume. We developed a novel microfluidic sonicator, which is able to achieve on-chip DNA/chromatin shearing into ideal fragment size (100~600bp) for both chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP). The integrated on-chip sonication followed by immunoprecipitation (IP) reaction can significantly reduce sample loss and contamination. Simple and accessible detection methods that can rapidly screen a large cell population with single cell resolution have been seriously lacking. We demonstrate a simple protocol for detecting translocation of native proteins using a common flow cytometer which detects fluorescence intensity without imaging. Using our approach, we successfully detected the translocation of native NF-kappa B (an important transcription factor) at its native expression level and examine the temporal dynamics in the process. Droplets with encapsulated beads and cells have been increasingly used for studying molecular and cellular biology. However, a mixed population of droplets with an uneven number or type of encapsulated particles is resulted and used for screening. We developed a fluorescence-activated microfluidic droplet sorter that integrated a simple deflection mechanism. By passing droplets through a narrow interrogation channel, the encapsulated particles were detected individually. The microcontroller conducted the computation to determine the number and type of encapsulated particles in each droplet and made the sorting decision. Our results showed high efficiency and accuracy for sorting and enrichment. / Ph. D.

A cell, the building block of all life, stores a plethora of information in its genome, epigenome, and transcriptome which needs to be analyzed via various Omic studies. The heterogeneity in a seemingly similar group of cells is an important factor to consider and it could lead us to better understand processes such as cancer development and resistance to treatment, fetal development, and immune response. There is an ever growing demand to be able to develop more sensitive, accurate and robust ways to study Omic information and to analyze subtle biological variation between samples even with limited starting material obtained from a single cell. Microfluidics has opened up new and exciting possibilities that have revolutionized how we study and manipulate the contents of the cell like the DNA, RNA, proteins, etc. Microfluidics in conjunction with Next Gen Sequencing has provided ground-breaking capabilities for handling small sample volumes and has also provided scope for automation and multiplexing. In this thesis, we discuss a number of platforms for developing low-input or single cell Omic technologies. The first part talks about the development of a novel microfluidic platform to carry out single-cell RNA-sequencing in a one-pot method with a diffusion-based reagent swapping scheme. This platform helps to overcome the limitations of conventional microfluidic RNA seq methods reported in literature that use complicated multiple-chambered devices. It also provides good quality data that is comparable to state-of-the-art scRNA-seq methods while implementing a simpler device design that permits multiplexing. The second part talks about studying the transcriptome of innate leukocytes treated with varying levels of LPS and using RNA-seq to observe how innate immune cells undergo epigenetic reprogramming to develop phenotypes of memory cells. The third part discusses a low-cost alternative to produce tn5 enzyme which low-cost NGS studies. And finally, we discuss a microfluidic approach to carrying out low-input epigenomic studies for studying transcription factors. Today, single-cell or low-input Omic studies are rapidly moving into the clinical setting to enable studies of patient samples for personalized medicine. Our approaches and platforms will no doubt be important for transcriptomic and epigenomic studies of scarce cell samples under such settings. / Doctor of Philosophy / This is the era of personalized medicine which means that we are no longer looking at one-size-fits-all therapies. We are rather focused on finding therapies that are tailormade to every individual’s personal needs. This has become more and more essential in the context of serious diseases like cancer where therapies have a lot of side-effects. To provide tailor-made therapy to patients, it is important to know how each patient is different from another. This difference can be found from studying how the individual is unique or different at the cellular level i.e. by looking into the contents of the cell like DNA, RNA, and chromatin. In this thesis, we discussed a number of projects which we can contribute to advancement in this field of personalized medicine. Our first project, MID-RNA-seq offers a new platform for studying the information contained in the RNA of a single cell. This platform has enough potential to be scaled up and automated into an excellent platform for studying the RNA of rare or limited patient samples. The second project discussed in this thesis involves studying the RNA of innate immune cells which defend our bodies against pathogens. The RNA data that we have unearthed in this project provides an immense scope for understanding innate immunity. This data provides our biologist collaborators the scope to test various pathways in innate immune cells and their roles in innate immune modulation. Our third project discusses a method to produce an enzyme called ‘Tn5’ which is necessary for studying the sequence of DNA. This enzyme which is commercially available has a very high cost associated with it but because we produced it in the lab, we were able to greatly reduce costs. The fourth project discussed involves the study of chromatin structure in cells and enables us to understand how our lifestyle choices change the expression or repression of genes in the cell, a study called epigenetics. The findings of this study would enable us to study epigenomic profiles from limited patient samples. Overall, our projects have enabled us to understand the information from cells especially when we have limited cell numbers. Once we have all this information we can compare how each patient is different from others. The future brings us closer to putting this into clinical practice and assigning different therapies to patients based on such data.