Roles of DNA Methyl Transferase 4 During Zebrafish Development

The research work presented in this dissertation describes tissue-specific functions of DNA methyltransferase 4 (an ortholog of the mammalian DNTM3B) during zebrafish hematopoiesis. Studies have implicated the involvement of DNA methylation in regulating hematopoiesis. Although DNA methylation by Dnmt1, Dnmt3a and Dnmt3b is required for HSC maintenance and self-renewal, the exact contributions of these enzymes in commitment of HSCs to different blood lineage precursors and terminal differentiation of these precursors into mature cells are not known. Answers to this idea are important as deregulations of DNMT1, DNMT3A and 3B are known to be involved in hematopoietic diseases. Thus, a better understanding of the contributions of DNMTs in regulating normal hematopoiesis will help in designing better drugs for treating hematopoietic diseases that arise due to aberrant methylation patterns. Chapter 1 is an introduction to DNA methylation and the enzymes, DNA methyltransferases that confer this methylation mark. This chapter summarizes literature from various model systems that allude to the requirement of DNA methyltransferases in a tissue-specific manner during normal development. Chapter 2 describes the utility of zebrafish as a model system to study DNA methylation. Also, I have characterized the embryonic expression patterns, iv morpholino knockdowns of DNA methyltransferases 4, 5, 6, 7, and 8 during zebrafish development. Chapter 3 discusses the epigenetic regulation of mature blood lineages by DNA methyltransferase dnmt4 in zebrafish. Here we show that dnmt4 is expressed in the hematopoietic compartment of zebrafish embryos. Transient knockdown of dnmt4 confers terminal differentiation defects of the erythroid and the myeloid cells. The data discussed in this chapter juxtapose dnmt4 morphants as an amenable model system to study the molecular pathways affected in (Immunodeficiency Centromere instability and Facial anomalies) ICF type-1 syndrome, which arises due to missense mutations in human DNMT3B. Chapter 4 describes the epistatic relationship of dnmt4 with the tumor suppressor gene apc in regulating zebrafish hematopoiesis. Chapter 5 presents the main conclusions of my dissertation work. I have described the implications of my findings and the future directions of this work.