Measuring Epigenetic Regulatory Network Dynamics

DNA encodes the functional units for the self-assembly of an embryo from a single cell to a complex multicellular organism. Yet how these units are assembled in space and in time depends on less well understood systems collectively termed epigenetic mechanisms. Historically, our ability to pinpoint critical genetic inputs to development has far outpaced our ability to dissect the epigenetic mechanisms underlying them. To build on our understanding of how embryos develop and to learn how to harness these systems for use in stem cell and regenerative therapies, we must understand comprehensively how epigenetic mechanisms choreograph the fundamental processes of embryogenesis. One major component of epigenetic systems is the organization of DNA in chromatin. Positioning of nucleosomes on DNA can effectively mask information encoded in the genome. However, over the period of development, the patterns of chromatin accessibility change due to the effects of epigenetic systems that drive both global and cell-type specific gains or losses in accessibility. By extension, the information in DNA available to the gene regulatory networks that pattern the embryo is constantly in flux. Understanding the drivers of epigenetic changes in chromatin accessibility is therefore critical to understand the flow of information through developmental networks. During development of the fruit fly Drosophila melanogaster, specialized transcription factors termed pioneer factors build the initial chromatin state in advance of the onset of embryonic patterning, and this initial state will sustain the first cell fate choices that the embryo will make. However, the initial chromatin state is only sufficient to sustain pattern formation to a point. Beyond such point, additional changes in the chromatin landscape must take place for development to proceed, and these changes are driven by as yet unidentified pioneers of cell-type specific chromatin states. I propose to measure how epigenetic complexity emerges from a relatively uniform ground state of chromatin structure by leveraging genetic manipulation of the Drosophila embryo with next-generation sequencing technologies. We will comprehensively identify the constellation of pioneer factors that install cell-type specific epigenetic signatures, and measure their recruitment to DNA in real time by a combination of ultrasensitive single-embryo genome-wide binding assays as well as a novel optical reporter system that we have developed.