Analysis of epigenetic gene regulation using a novel zebrafish epigenetic reporter transgenic line

Embryonic development, a process in which pluripotent cells differentiate into specific organs, is controlled by genetic and epigenetic factors. Epigenetic mechanisms, including DNA methylation, histone modifications, chromatin remodeling, and others, play crucial roles in this process. Human epigenome sequencing projects and several additional studies revealed that tissue specific epigenetic marks specify cell identity and tissue specific gene expression during development. Remarkably, the process is highly conserved between vertebrates. Large-scale genetic screens performed in fruit flies and nematode worms have been very successful in identifying epigenetic regulators in invertebrates. However, comparable screens have not been carried out in vertebrates, and mechanisms of tissue-specific epigenetic regulation in vertebrates are still not well understood. We have developed a novel transgenic epigenetic reporter zebrafish line that reliably reports changes in tissue specific epigenetic silencing based on the dynamic expression of destabilized green fluorescent protein (GFPd2). Using this line, we are performing the first ever large scale ENU mutagenesis F3 genetic screen in a vertebrate to identify recessive mutants that regulate epigenetic silencing or activation. One of the mutants isolated through the forward genetic screen contains a mutation in a largely uncharacterized histone modifying gene. Preliminary analysis of the mutant phenotypes revealed that the mutated gene likely plays crucial roles in neural tissue development. Through morpholino silencing of the gene, we phenocopied the abnormal head morphology and epigenetic silencing of the brain in the ENU mutant, verifying the mutation induces a loss-of-function phenotype. We are now comprehensively characterizing the roles and epigenetic modifications regulated by this gene.