It is well established that epigenetics and chromatin modifications are important factors that can govern gene activity and nuclear architecture. They are also proven to be essential for normal embryonic development and cell differentiation. One important event during mouse development is the establishment of epigenetic reprogramming which is believed to be essential for normal growth and development, however; the mechanism is still poorly understood. The general objective of this PhD study was to investigate the profiles and mechanisms of epigenetic and chromatin modifications during normal mouse development and in embryonic stem cells. Mouse pre- and postimplantation embryos and ES cells were used in experiments employing a range of different methodologies. The dynamics of epigenetic DNA and histone methylation were captured using laser confocal immunofluorescent microscopy and western blotting. The activity of epigenetic modifiers was monitored by real-time PCR and candidate genes were validated using siRNA technology. The present studies demonstrate that heterochromatin markers H3K9me3, H3K9me2, H4K20me2, H4K20me3, HP1α and HP1β are reprogrammed during early development. Demethylation of H3K9me2, H3K9me3 and H4K20me3 took place at two-cell stage and remethylation occurred at four-cell stage except for H4K20me3. The reestablishment of H4K20me3 was initially observed in early postimplantation embryos in extraembryonic tissue, specifically in the mural trophectoderm. In embryonic tissue, H4K20me3 was not clearly detected until in mid to late postimplantation development. The mechanism of H3K9me2 and H3K9me3 demethylation might be due to either an imbalance of epigenetic modifiers or the presence of Jmjd2a and Jmjd1a histone demethylase postfertilisation. We have also report evidence that HP1α and Suv4-20h are required in heterochromatin before the recruitment of H4K20me3 during mouse development and in ES cells. Therefore H4K20me3 removal was believed to involve the lack of prerequisite heterochromatin complexes such as HP1α and Suv4-20h enzymes. Furthermore, the presence and levels of H4K20me3 and HP1α might be strongly associated with cell differentiation and tissue maturation in mouse in vivo development but not in vitro early differentiated ES cells. Surprisingly, the results showed that chromatin modifications and their modifiers in ES cells are different from ICM and epiblast. Chromatin modifications H4K20me3 and HP1α were absent from ICM and epiblast, but were detected in ES cells. Notably, H4K20me3 and HP1α were established after early incubation of ICM into ES cell medium, but this change was not dependent on the presence of serum and leukaemia inhibiting factor. Epigenetic modifier Jmjd2a but not Jmjd1a was found in ICM. Conversely, Jmjd1a is highly expressed in ES cells while Jmjd2a was inactivated. In addition, the present studies revealed the substantial role of histone demethylases in development, as it may be important for epigenetic reprogramming. The results demonstrated that inhibition of demethylase Jmjd2a and Jmjd1a caused preimplantation embryos to arrest at the twocell stage while Jmjd2c deficient embryos failed to reach blastocyst. Thus it is possible that Jmjd2a and Jmjd1a were essential for epigenetic reprogramming while Jmjd2c is critical for cell fate establishment during blastocyst formation. In conclusion, the global chromatin signature in ES cells differs from ICM and epiblast; heterochromatin reprogramming occurs at two-cell stage; maturation of heterochromatin occurs at postimplantation; and histone demethylases Jmjd1a, Jmjd2a and Jmjd2c are important in preimplantation development. Results from the present studies could provide crucial information for developmental biology and stem cell research, and provide as a model for improvement of reproductive biotechnologies such as somatic cell reprogramming, and diagnosis of epigenetic abnormalities in early development.

Publicly-available microarray data constitutes a huge resource for researchers in biological science. A wealth of microarray data is available for the model organism – the mouse. Pluripotent embryonic stem (ES) cells are able to give rise to all of the adult tissues of the organism and, as such, are much-studied for their myriad applications in regenerative medicine. Fully differentiated, somatic cells can also be reprogrammed to pluripotency to give induced pluripotent stem cells (iPSCs). ES cells progress through a range of cellular states between ground state pluripotent stem cells, through the primed state ready for differentiation, to actual differentiation. Microarray data available in public, online repositories is annotated with several important fields, although this accompanying annotation often contains issues which can impact its usefulness to human and / or programmatic interpretation for downstream analysis. This thesis assembles and makes available to the research community the largest-to-date pluripotent mouse ES cell (mESC) microarray dataset and details the manual annotation of those samples for several key fields to allow further investigation of the pluripotent state in mESCs. Microarray samples from a given laboratory or experiment are known to be similar to each other due to batch effects. The same has been postulated about samples which use the same cell line. This work therefore precedes the investigation of transcriptional events in mESCs with an investigation into whether a sample's cell line or source laboratory is a greater contributor to the similarity between samples in this collected pluripotent mESC dataset using a method employing Random Submatrix Total Variability, and so named RaSToVa. Further, an extension of the same permutation and analysis method is developed to enable Discovery of Annotation-Linked Gene Expression Signatures (DALGES), and this is applied to the gathered data to provide the first large-scale analysis of transcriptional profiles and biological pathway activity of three commonly-used mESC cell lines and a selection of iPSC samples, seeking insight into potential biological differences that may result from these. This work then goes on to re-order the pluripotent mESC data by markers of known pluripotency states, from ground state pluripotency through primed pluripotency to earliest differentiation and analyses changes in gene expression and biological pathway activity across this spectrum, using differential expression and a window-scanning approach, seeking to recapitulate transcriptional patterns known to occur in mESCs, revealing the existence of putative “early” and “late” naïve pluripotent states and thereby identifying several lines of enquiry for in-laboratory investigation.

No description available.

TLE proteins are a closely related family of vertebrate corepressors. They have no intrinsic DNA binding ability, but are recruited as transcriptional repressors by other sequence specific proteins. TLE proteins and their homologues in other species have been implicated in many developmental processes including neurogenesis, haematopoiesis and the formation of major organs. They have also been implicated in early lineage specification in vertebrates but a direct role in this has not been found in mammals. The aim of my PhD is therefore to analyse the function of TLE proteins in early lineage specification and cell fate decisions using mouse embryonic stem cells (ESCs) as a model. The investigation of this has previously been complicated, firstly by the large array of transcription factors that TLEs interact with and secondly by redundancy between similar TLE proteins hindering loss of function approaches. To circumvent these problems, I have used two complementary experimental strategies. The first was identification of point mutations in TLE1 that affect specific classes of DNA binding. Two of these mutations L743F and R534A were of particular interest and were reversibly overexpressed in ES cells to correlate phenotypes to biochemical activity. The second strategy was the mutation of the two primary TLC genes in ES cells and early mouse embryos, TLE3 and TLE4. Complementary evidence from these approaches revealed a role for TLEs in the promotion of ES cell differentiation by repression of pluripotency/self-renewal associated genes. Additionally, neural specification was increased by TLE1 expression especially by the TLE1 point mutations, highlighting opposing roles for negative effects on mesendodermal differentiation. Early mesoderm/primitive streak was increased by loss of TLE, probably through Wnt antagonism. Anterior endoderm was increased by reduced TLE, but a critical level of TLE was still necessary and TLE1 overexpression also upregulated some anterior endoderm markers suggesting both negative and positive roles for TLE proteins in this process.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18183号 / 医博第3903号 / 新制||医||1004(附属図書館) / 31041 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 髙橋 良輔, 教授 髙橋 淳, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM