As described in the goals and objectives section of this report, this project consists of three specific aims: Role of Dnmt3bb.1 in hematopoiesis and eye development Previously, we showed that Dnmt3bb.1 mediates hematopoietic stem and progenitor cell (HSPC) fate maintenance in zebrafish embryos. We reported that dnmt3bb.1 is specifically targeted to the gene body region of the cmyb locus, and that it positively regulates expression of this essential transcription factor to maintain hematopoietic specification. More recently, we have also reported that Dnmt3bb.1 place a key role in eye development as well, using zebrafish and another useful fish model for studying epigenetic gene regulation, the Mexican tetra (Astyanax mexicanus). Astyanax mexicanus is a freshwater fish native to Mexico. A few thousand years ago some of these fish were trapped in dark caves, and over the years they evolved into a completely different cave morph (referred as cavefish) that lacked eyes and pigmentation and acquired several other key adaptations such as extra sensory hair cells, metabolic adaption to food scarcity, and neuronal alterations. Since cavefish and surface (eyed) morphs very recently, they can interbreed and share near identical genomes. Although previous studies identified eye development genes associated with the loss of eyes phenotype in the Pachn blind cavefish morph of Astyanax mexicanus, no inactivating mutations have been found in any of these genes. We have found that excess DNA methylation-based epigenetic silencing due to increased Dnmt3b promotes eye degeneration in cavefish. By performing parallel analyses in cavefish and surface morphs of Astyanax and in the zebrafish Danio rerio, we have discovered that DNA methylation mediates eye-specific gene repression and globally regulates early eye development. The most significantly hypermethylated and down-regulated genes in the cave morph are also linked to human eye disorders, suggesting the function of these genes is conserved across the vertebrates. Our results show that changes in DNA methylation-based gene repression can serve as an important molecular mechanism generating phenotypic diversity during development and evolution. We are currently following up on these studies to better understand the role of DNA methylation in both hematopoiesis and eye development. Epigenetic regulation of metabolism In addition to eye and pigment loss and other adaptations, Astyanax cavefish have extreme and unusual metabolic adaptations that allow them to survive chronic and long-term food deprivation. These include excess fat deposition, altered liver function, and resistance to metabolic disease. We hypothesize that in a similar manner to loss of eyes, changes in epigenetic gene regulation may also underlie cavefish metabolic adaptations. We are using single-cell profilingto investigate differences in adipocytes and other cell types in the muscles (where there is large amounts of fat stored in cavefish) and livers of cavefish and surface fish. We are also performing whole genome bisulfite sequencing and RNAseq from surface and cavefish muscles and livers to identify differentially expressed and methylated genes. Preliminary results from bisulfite sequencing and marker analysis suggest that key fat metabolism genes such as ucp1 and ucp2 are differentially expressed and differentially methylated in cavefish liver and muscles respectively. We will follow up on these findings to elucidate how differential DNA methylation influencesd fat metabolism and obesity. Isolation and characterization of tissue-specific epigenetic regulators Genetic screens carried out in Drosophila and C. elegans have been highly successful in identifying genes regulating cell-type specific epigenetic gene regulation in invertebrates, but the molecular mechanisms involved in organ- and tissue-specific epigenetic regulation in vertebrates are still relatively unknown. We have developed a novel zebrafish transgenic reporter line that allows us to monitor dynamic changes in DNA methylation-based epigenetic regulation in intact animals during development. Using this transgenic line, we are performing the first large-scale F3 genetic screen in a vertebrate to identify recessive mutants in regulators of epigenetic gene silencing or activation. A pilot screen of fifty F2 families has already yielded eleven mutants defective in ubiquitous or tissue-specific epigenetic gene silencing or activation. The isolated mutants show a wide array of phenotypes ranging from complete lack of GFP expression to gain or loss of GFP expression in specific organs such as liver, brain, blood cells and eyes. Using RNAseq-based mapping, we have already successfully mapped three mutants and are actively performing additional work to conclusively identify the mutated genes. The identification and functional characterization of the mutated genes from our ongoing genetic screen is likely to yield many important and valuable new insights into epigenetic regulation in vertebrates, just as comparable powerful genetic screens carried out in invertebrates have done.
