Epigenetics of Development

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

Investigators

Linked publications & trials

Abstract

As described in the goals and objectives section of this report, this project consists of three specific aims: 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 EpiTag 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 have performed the first large-scale F3 genetic screen in a vertebrate to identify recessive mutants in regulators of epigenetic gene silencing or activation, yielding a number of 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 most of these mutants and are actively performing additional work to conclusively identify and further characterize the mutated genes. These genes include a novel member of a well-characterized family of histone modifying enzymes that we have identified as the first known epigenetic regulator of arterial endothelial identity. Further functional characterization of the mutated genes identified in 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. Epigenetic regulation of regeneration and reproduction In addition to serving as a highly effective reporter for our successful genetic screen for epigenetic regulators, we also discovered that the EpiTag line shows a remarkable strong activation in, and serves as a superb marker for, cells at early stages of gametogenesis and regenerating cells in the fin, heart, and trunk muscle. Our working hypothesis is that EpiTag expression marks very early stages where epigenetic reprograming is taking place in both contexts. We are pursuing follow-up studies on these observations as well. We will initially use the well-studied fin regeneration model for two focused questions related to regeneration. A variety of evidence points to the importance of DNA methylation and other epigenetic changes during zebrafish fin regeneration. However, other recent work has suggested that global DNA methylation does not change dramatically during regeneration, although these studies rely on samples containing mixed populations of regenerating cells at different stages as well as non-regenerating cells. We will (i) determine what changes are specifically associated with the earliest EpiTag+ stages of regeneration by transcriptomic and epigenetic profiling of EpiTag+ cells isolated from regenerating fins. Although the blastema was historically thought to be derived from a stem-like multipotent cell population, recent studies have shown that lineage-restricted cells retaining memory of their original identity contribute to regeneration in zebrafish, salamanders, and even mice. However, it is still unclear precisely which cells contribute to regeneration and how. We will (ii) examine the identity of cells cycling through the early EpiTag+ stages of regeneration by performing scRNAseq at multiple regenerating stages to determine what cells contribute to EpiTag+ cell clusters, what they become, and how they relate to cell types in non-regenerating fins. Epigenetic reprogramming takes place in the mammalian germline via global erasure of DNA methylation. However, recent studies suggest that similar genome-wide erasure of DNA methylation does not take place in the zebrafish germline, although this work was carried out on mixed populations of vasa:gfp+ cells at different stages of germ cell development rather than cells at specific defined stages of gametogenesis from adult gonads, potentially masking transient or more limited changes. We are validating early stages of gametogenesis marked by EpiTag reporter expression using specific IHC with markers and scRNAseq , and profiling the transcriptomic and epigenetic landscape of these EpiTag+ cells. Our working hypothesis is that at least limited epigenetic changes occur during EpiTag+ early gametogenesis. Epigenetic changes in cavefish adaptation and evolution 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 profiling to 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 influences fat metabolism and obesity.