Epigenetic and chromatin reprogramming in mouse development and embryonic stem cells
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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.