The roles of DPPA2, DPPA4 and SMARCA5 in mouse zygotic genome activation, epigenetic reprogramming and development
The epigenetic remodelling that takes place in order to transform a fertilised oocyte into an embryo and then a whole organism, is one of the most intriguing cellular transformations in biology. The whole process starts with the meiotic resumption of an oocyte when the translational activation of the dormant mRNAs takes place, followed by their gradual elimination in the zygote, and ending with the full transcriptional activation of the newly reprogrammed embryonic genome. All these complex processes constitute the maternal to zygotic transition (MZT), which is accompanied by the global epigenetic reprogramming of the embryo. In this dissertation, I focus predominantly on the epigenetic factors that potentially contribute to the initiation of transcription, a process also called zygotic genome activation (ZGA). In mice, this takes place in 1 cell embryos – minor ZGA, followed by a burst of transcription - major ZGA, at the 2 cell stage. Previous screens in the lab, using mouse embryonic stem cell (mESC) identified the small DNA binding proteins DPPA2, DPPA4 and the chromatin remodeler SMARCA5 as potential inducers of major ZGA. Here I focus on validating these three factors and I assess their roles in vivo, using conditional knock-out mouse models and a targeted protein depletion system, together with the characterisation of the transcriptomic and epigenetic changes. In Chapter 1, I give an overview of the biological context for my study together with the latest findings, and depict some of the technologies used to describe the changes that take place during epigenetic reprogramming in the early embryo. Chapter 2 outlines the materials and methods used to address my questions, and Chapters 3, 4 and 5 contain a detailed description of my results and their significance in the wider context. Chapter 3 is focused on the definition of comprehensive ZGA gene lists and of control genes, building a base for chapters 4 and 5. Using six published transcriptomics datasets from independent studies using different library generation methods, I define minor and major ZGA signatures that give a complex picture of the transcriptional landscape in zygotes and 2 cell stage embryos, by waning the biases introduced with each individual study. In Chapter 4, I assess the role that maternal DPPA2 and DPPA4 play in mouse embryonic development and major ZGA. For this, I used single and double conditional knock-out models to deplete maternal deposits of these proteins, and showed that major ZGA still takes place in their absence. This work was recently published. In Chapter 5, I assess the role that the maternal ISWI ATPase SMARCA5 plays in embryonic development, ZGA and chromatin remodeling in early embryos. For this I use both a conditional knock-out mouse model lacking SMARCA5 in oocytes, but also a targeted protein depletion system in early wild-type zygotes, followed by transcriptomics, methylation and chromatin accessibility characterisation of the embryos. Both systems confirmed that SMARCA5 plays an important role not only in the transcriptional activation but also in global chromatin changes that take place at the 2 cell stage. Chapter 6 contains a summary of my findings, conclusions and their relevance in the wider context of the field, and ends with proposed future directions. My dissertation provides new insights into the mechanisms and factors that regulate mouse ZGA, and highlights the importance of validating in vitro findings in a relevant in vivo system.
ZGA, mouse development, zygotic genome activation, smarca5, dppa2, dppa4
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