Project Summary: The developmental program encodes mechanisms that shape the chromatin landscape and drive large-scale transitions in its organization, but the mechanisms whereby these changes are integrated into embryonic pat- terning systems remain unclear. The genetic program that directs the initial patterning of the Drosophila embryo has been exhaustively characterized, and many of the cis-regulatory elements whereby transcription factors pat- tern the embryo have been identified. However, only recently has the field been able to measure changes in chro- matin accessibility and occupancy with sufficient sensitivity to observe that these mechanisms are dynamic and play critical yet underappreciated roles in development. Cis-regulatory elements are not static in their chromatin state but instead change accessibility and modification status as a function of progression through the develop- mental program. Interfering with the temporal progression of chromatin states disrupts embryonic patterning. This project seeks to identify how patterning systems choreograph changes in epigenetic state to further under- stand how this contributes to the regulatory logic of embryogenesis. The objective of this project is to evaluate the impact of regulated chromatin states on the operation of a model gene regulatory network critical for Dro- sophila segmentation, and to investigate in detail the mechanism whereby one component of the network pio- neers open chromatin states. The central hypothesis is that regulatory networks employ transcription factors whose primary role is to modulate accessibility states, and that this enables networks to generate more complex patterns from a limited set of input factors. This project is justified through the rationale that the Drosophila system provides unmatched resources for performing time-resolved genome-wide measurements of transcrip- tion factor occupancy and chromatin accessibility in addition to allowing for the genetic manipulation of these components for the comprehensive examination of dynamic chromatin states in the context of a developmentally relevant regulatory network. The first aim will determine through the use of time-resolved ChIP- and ATAC-seq on wild-type and mutant embryos the influence of chromatin accessibility on the binding site selection for all transcription factors operating within a model gene regulatory network. The second aim will focus through the use of a combination of biochemical, genetic, and genomic approaches on the molecular mechanism for pioneer- ing chromatin accessibility by one component of the network. The third aim will investigate through the use of genetics and genomics how the embryo determines a period of competence for certain regulatory elements to be granted accessibility. The significance of this proposal stems from the innovation to further the understanding of epigenetic mechanisms of gene regulation within the broader framework of regulatory networks in develop- me...